Rajagopal, Mogana Sundari (2014) Canarium patentinervium miq....

CANARIUM PATENTINERVIUM MIQ.

(BURSERACEAE KUNTH.):

A PHYTOCHEMICAL AND

PHARMACOLOGICAL STUDY

MOGANA SUNDARI RAJAGOPAL

B. Pharm. (Hons)

THESIS SUBMITTED TO THE UNIVERSITY OF

NOTTINGHAM (MALAYSIA CAMPUS) FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

AUGUST 2014

ii

DECLARATION

I, R.Mogana, declare that this thesis is my own work. It is being submitted for the

Degree of Doctor of Philosophy, at the School of Pharmacy, Faculty of Sciences,

University of Nottingham, Malaysia. It has not been submitted before for any

degree or examination at this or any other University.

«««««

Signature

«««««

Date

iii

DEDICATION

To my beloved Sri Sri Radha Krsna, my beloved parents, G. Rajagopal

and P. Muthuletchumy and my beloved husband P. Ramesh Kumar

iv

ACKNOWLEDGEMENTS

.

I offer my greatest thanks to my dear husband and beloved parents for their love,

ongoing support and motivation.

I am grateful to my supervisor Associate Professor Dr.Christophe Wiart for his

constant enthusiasm and commitment, as well as his support all through the course of

this research. Thank you for inspiring me and for the steadfast encouragement in the

field of phytochemistry and emotional support. You have been a great inspiration.

Thank you!

I offer thanks to my co-supervisor Dr. Khoo Teng Jin for his kind attention and

guidance. I would also like to thank Dr Achyut Adhikari for his valuable expertise in

NMR. In addition I would also like to thank Dr.Tracey Bradshaw on her assistance

with the anticancer work, Dr Banasri Hazra on her assistance with the antileishmanial

and Assoc. Prof. Nongyao Sawangjaroen for the assistance on the parasitic work.

v

ABSTRACT

Canarium patentinervium Miq. belongs to the family of Burseraceae best known for

producing resins of economic, medicinal, and cultural values such as frankincense,

myrrh, and copal. This family consists of 18 genera and 700 species of trees. In the

Asia-Pacific region, about 20 species of Burseraceae are used to treat haemorrhoids,

heal wounds and to treat skin infections. This plant has been used to heal wounds

amongst the indigenous people of Malaysia. Furthermore no pharmacological and

phytochemical studies have been reported on this species. This study was undertaken

to screen the phytochemical and pharmacological aspects of this plant. Qualitative

phytochemical properties of the crude extract was determined for the presence of

tannins, flavonoids, alkaloids, saponins or sterols. Phytochemical analysis of

Canarium patentinervium Miq. revealed presence of tannins and flavonoids in the

ethanol extract of leaves and barks. Bioassay guided fractionation led to isolation of

eight secondary metabolites by chromatography which were identified by NMR

techniques. Two coumarins (scopoletin and scoparone), five phenols (cynaroside,

hyperin, (+)-catechin, lioxin and syringic acid) and a norsesquiterpene with

cyclohexenone ring (vomifoliol). The latter three compounds were isolated for the

first time from the genus Canarium. The plant and the isolated compounds were then

subjected to six biological assays comprising of antibacterial, antioxidant, anticancer,

anti-inflammatory, anti-acetylcholinesterase and anti-parasitic activities. Infectious

diseases remain the leading cause of death worldwide and bacteria have become more

resistant to conventional antibiotic and the search for novel antimicrobial agents from

medicinal plants has become crucial. Antibacterial test was done using disc diffusion

method, minimum inhibitory concentration (MIC), minimum bactericidal

vi

concentration (MBC) and death kinetic assay with ampicillin as the positive control.

The ethanol extract of leaves and the hexane extract of bark displayed remarkable

antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria.

All isolated compounds tested against S.aureus ATCC 11632 showed bacterial

growth inhibition. Scopoletin, scoparone, hyperin, cynaroside and syringic acid had

bactericidal effect <100 µg/ml. Only scopoletin had bactericidal effect and complete

kill at MBC 50.00 µg/ml. Bacterial infections have been known to generate extensive

formation of free radicals which is becoming increasingly recognized in the

pathogenesis of the many human diseases. The role of free radicals and active oxygen

is becoming increasingly recognized in the pathogenesis of the many human diseases,

including cancer, neurodegenerative diseases, ageing, and atherosclerosis. Five

various antioxidant assays with different mode of action [2, 2-diphenyl-1-

picrylhydrazyl (DPPH) and 2, 2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)

(ABTS), ferric reducing antioxidant power (FRAP), ȕ-carotene bleaching assay and

superoxide dismutase (SOD) assay] were used to test the antioxidant scavenging

abilities of this plant. Vitamin C (L-ascorbic acid), quercetin and trolox were used as

positive controls. The ethanol extract of leaves and barks displayed superior

antioxidant capacities. The EC50 values of the samples were consistently low in SET

methods (ABTS, DPPH and FRAP) superior to standard as opposed to HAT method

ȕ-carotene bleaching assay). Hyperin and (+)-catechin exhibited the most consistent

free radical scavenging capability across the five antioxidant assay. Hyperin and (+)-

catechin have significantly lower IC50 (0.75±0.03 µg/ml and 0.94±0.27 µg/ml

respectively) compared to SOD enzyme (IC50 1.59±04 µg/ml). Scopoletin exhibited

potent antioxidant activity compared to scoparone with significantly lower EC50

values in ABTS (IC50 1.08±0.03 µg/ml) compared to ascorbic acid (EC50 1.54±0.03

vii

µg/ml) and lower values in FRAP assay (FRAP value 49.00±0.64 µg/ml) than

quercetin (FRAP value 86.00±0.24 µg/ml) and ascorbic acid (FRAP value

347.00±0.23 µg/ml). 9RPLIROLRO KDG SRWHQW ȕ-carotene bleaching activity with IC50

6.85±0.37 µg/ml. Infections and free radical generation are recognized in the

pathogenesis of cancer. The ethanol and chloroform extract of barks showed

significant anticancer activities with GI50 values of 34.40µg/ml and 23.44µg/ml. The

most susceptible cell lines were found to be the breast cancer cell line, MDA 468.

Scopoletin displayed potent anticancer effect against breast cancer cell line MDA 468

(GI50 0.09±0.25 µg/ml) and colorectal cancer cell line HT-29 (GI50 0.17±0.05 µg/ml),

the latter being more significant that positive control doxorubicin (GI50 0.66±0.60

µg/ml). In recent years, roles have been identified for several inflammatory cells and

for a large number of inflammatory mediators in important pathologies not previously

linked to inflammation, such DV $O]KHLPHU¶V GLVHDVH DQG FDUGLRYDVFXODU GLVRUGHUV

including atherosclerosis, as well as cancer. Recently, reports have appeared

regarding so-FDOOHG ³GXDO LQKLELWRUV´ DJHQWV WKDW LQKLELW QRW RQO\ cyclooxygenase-1

(COX-1) and cyclooxygenase-2 (COX-2), but also 5-lipoxygenase (5-LOX).

Chloroform extract of the barks had the lowest 5-LOX inhibition (IC50=29.53±0.0

ȝJPO ZKHQ FRPSDUHG WR 1'*$ (IC50= ȝJPO Ethanol extract of

leaves had superior COX-1 inhibition (IC50 = 0.60±0.01µg/ml) compared to COX-2

inhibition (IC50 = 1.07±0.01 µg/ml), whereas the barks had superior COX-2 inhibition

(IC50 = 9.39±0.03 µg/ml) as opposed to COX-1 (IC50 = 11.41±0.03 µg/ml). All

isolated compounds exhibited significantly lower 5-LOX inhibition than NDGA.

Scopoletin and scoparone were potent inhibitors of 5-LOX recording lowest IC50

values (IC50 0.34±0.01 µg/ml and 0.20±0.01 µg/ml respectively). However (+)-

catechin had a more comprehensive anti-inflammatory activity with dual inhibition of

viii

5-LOX (IC50 16.10±0.03 µg/ml) and COX (COX-1; IC50 12.08±0.02 µg/ml, COX-2;

IC50 83.89±0.03 µg/ml). Syringic acid exhibited potent 5-LOX inhibition (IC50

1.38±0.03 µg/ml) and moderate COX-1 inhibition (IC50 34.89±0.02 µg/ml).

Furthermore, oxidative and inflammatory processes are among the pathological

features associated with the central QHUYRXVV\VWHPLQ$O]KHLPHU¶VGLVHDVH7KHUH LV

evidence that acetyl cholinesterase (AChE) inhibitors have an anti-inflammatory role

through action against free radicals and amyloid toxicity, as well as through

decreasing release of cytokines from activated microglia in the brain and blood.

Chloroform extract of the barks displayed the best activity (IC50 ȝJPO

as opposed to positive control, galanthamine (IC50 = 0.74±0.ȝJPO7KHHWKDQRO

extract of barks and leaves follow through with IC50 ȝJPODQG,&50 =

ȝJPOUHVSHFWLYHO\. Only scopoletin, scoparone, vomifoliol and syringic

acid showed AChE inhibition at IC50 <100 µg/ml. Syringic acid exhibited good AChE

inhibition (IC50 29.53±0.19 µg/ml), lowest of all compounds tested. Choline is the

precursor of phosphatidylcholine (PC), a main component of Leishmania

promastigote membranes. Therefore, inhibition of choline formation may decrease

Leishmania survival. This hypothesis can be tested by using inhibitors of the

acetylcholinesterase enzyme (AChE), which catalyzes the hydrolysis of acetylcholine

to choline and acetic acid, as leishmanicidal compounds. The hexane extract of leaves

showed moderate antileshmanial activity with IC50 YDOXHVRIȝJPO2QO\

ethanol extracts showed activity against Giardia intestinalis and Entamoeba

histolytica DW FRQFHQWUDWLRQ RI ȝJPO 6copoletin was tested against all three

parasite amd it was more potent against Leishmania donovani (IC50 163.30±0.32

µg/ml) and MIC of >200 µg/ml for both Giardia intestinalis and Entamoeba

histolytica. Six kinds of major biological effects were evident in the crude and

ix

compounds namely, antioxidant, antibacterial, anti-inflammatory, anti-AChE, anti-

parasitic, and anticancer all of which were reported for the first time from this plant.

Given the aforementioned evidence it is tempting to speculate that Canarium

patentinervium Miq. represents an exciting scaffold from which to develop leads for

treatment of inflammatory and oxidative stress related diseases.

x

PUBLICATIONS ARISING FROM THIS STUDY*

x R. Mogana, K. Teng--LQDQG&:LDUW³,Q9LWUR$QWLPLFURELDO$QWLR[LGDQW

Activities and Phytochemical Analysis of Canarium patentinervium Miq. from

0DOD\VLD´ %LRWHFKQRO 5HV ,QW YRO S (abstract pg

377).

x R. Mogana, T. D. Bradshaw, T. J. KhRRDQG&:LDUW³,Q9LWUR$QWLWXPRU

potential of Canarium patentinervium 0LT´$FDG-&DQFHU5HVYROQR

1, pp. 1±4, 2011 (abstract pg 378).

x R. Mogana DQG & :LDUW ³Canarium / ௗ $ 3K\WRFKHPLFDO DQG

3KDUPDFRORJLFDO5HYLHZ´-3KDUP5HVYRO. 4, no. 8, pp. 2482±2489, 2011

(abstract pg 379).

x A Nematollahi , N Aminimoghadamfarouj , M Rajagopal , TJ Khoo, C.Wiart,

³7KH ILUVW DQWLEDFWHULDO DFWLYLW\ UHSRUW RI WKUHH VHOHFWHG0DOD\VLDQ UDLQIRUHVW

PHGLFLQDOSODQWV´3ODQWD0HGYRO±PL9, 2011 (abstract pg 380).

x R. Mogana DQG & :LDUW ³$QWL-Inflammatory, Anticholinesterase, and

Antioxidant Potential of Scopoletin Isolated from Canarium patentinervium

0LT%XUVHUDFHDH.XQWK´(YLGHQFH-Based Complement. Altern. Med., vol.

2013, p. 734824, 7 pages, 2013 (abstract pg 381).

x R. Mogana, K. Teng--LQ DQG &:LDUW ³7KH0HGLFLQDO 7LPEHUCanarium

patentinervium Miq. (Burseraceae Kunth.) Is an Anti-Inflammatory

Bioresource of Dual Inhibitors of Cyclooxygenase (COX) and 5-Lipoxygenase

(5-/2;´,651%LRWHFKQRl., vol. 2013, pp. 1±8, 2013 (abstract pg 382).

*Appendix C

xi

CONFERENCE PROCEEDINGS AND TALKS RELATED TO

THIS STUDY

x Oral presentation at the 25th Scientific Meeting of the Malaysian Society of

Pharmacology & Physiology, 25th-26th of May 2011 at Faculty of Medicine &

Medical Sciences, Universiti Putra Malaysia. ± ³Canarium- a source of new

DQWLELRWLFVDQGDQWLR[LGDQWV"´

x Poster presentation at the 59th International Congress and Annual Meeting

of the Society for Medicinal Plant and Natural Product Research, 4 ± 9th

September 2011, Maritim Pine Beach Resort Hotel Antalya, Turkey. ³The

first antibacterial activity report of three selected Malaysian rainforest

PHGLFLQDOSODQWV´

x Poster presentation at the International Conference on Natural Products, 13-

16th November 2011, Palm Garden IOI Resort, Putrajaya, 0DOD\VLD³In vitro

antimicrobial, antioxidant, anticancer activities and phytochemical

analysis of Canarium patentinervium Miq. From Malaysia ³

x Oral presentation at the International Conference on Natural Products, 4-6th

March 2013, Shah Alam Convention Centre0DOD\VLD ³Antiinflammatory,

anticholinesterase and antioxidant activities of scopoletin isolated from

Canarium VS%XUVHUDFHDH.XQWK´

x Oral presentation at the Graduate School Talk, 27th June 2012, University of

Nottingham (Malaysia Campus). ³Canarium patentinervium Miq.: The

SK\WRFKHPLFDODQGSKDUPDFRORJLFDOVWXG\´.

xii

LIST OF FIGURES

Page

Figure 1.1: Chemical structures of secondary metabolites from Canarium

L.

24

Figure 1.2: A diagrammatic summary of steps in the study of Canarium

patentinervium Miq. evaluating the phytochemistry and

pharmacological activities.

46

Figure 2.1: Fresh and dried herbarium sample of Canarium

patentinervium Miq.

48

Figure 3.1: Positive flavonoid result for leaf ethanol extract (left) against

blank and and bark ethanol extract (right) against blank.

55

Figure 3.2: Positive tannin result for leaf ethanol extract marked as test

WXEHµ/(¶OHIWDQGEDUNHWKDQROH[WUDFWPDUNHGDVWHVW

WXEHµ6¶ULJKWDJDLQVWEODQN-), positive standard tannic

acid(+) and ferric chloride.

56

Figure 3.3: Positive sterol test for leaf ethanol extract (left) and bark

ethanol extract (right) against blank.

57

Figure 3.4: Positive sterol test for bark hexane extract (left) and bark

chloroform extract (right) against blank.

58

Figure 4.1: Diagrammatic representation of chemical reaction of the

reaction of DPPH in the presence of an electron donating

antioxidant.

67

Figure 4.2: Diagrammatic representation of the formation of the ABTS

radical after the addition of potassium persulphate.

70

xiii

Figure 4.3: Formation of (Fe2+-TPTZ) complex from (Fe3+-TPTZ)

complex by antioxidant

73

Figure 4.4: )RUPDWLRQRIDGGXFWVIURPE\ȕ-carotene and antioxidant

with a lipid peroxide radical.

75

Figure 4.5: DPPH test result with 96 well microtiter plate results for bark

(left) and leaf (right) ethanol extract.

81

Figure 4.6: DPPH scavenging activity (%) of Canarium patentinervium

Miq.

83

Figure 4.7: ABTS scavenging activity (%) of Canarium patentinervium

Miq.

84

Figure 4.8: Fe (II) calibration curve in FRAP assay. 87

Figure 4.9: Leaf hexane (LH) extract calibration curve in FRAP assay. 88

Figure 4.10: Leaf chloroform (LC) extract calibration curve in FRAP

assay.

89

Figure 4.11: Leaf ethanol (LE) extract calibration curve in FRAP assay. 90

Figure 4.12: Bark hexane (BH) extract calibration curve in FRAP assay. 91

Figure 4.13: Bark chloroform (BC) extract calibration curve in FRAP

assay.

92

Figure 4.14: Bark ethanol (BE) extract calibration curve in FRAP assay. 93

Figure 4.15: Ascorbic acid (AA) calibration curve in FRAP assay. 94

Figure 4.16: Trolox (TRO) calibration curve in FRAP assay. 95

Figure 4.17: Quercetin (QC) calibration curve in FRAP assay. 96

Figure 4.18: ȕ-carotene decolourisation (%) of Canarium patentinervium

Miq.

97

Figure 4.19: Gallic acid calibration curve in Folin-Ciocalteu assay. 98

xiv

Figure 4.20: Quercetin calibration curve in total flavonoid content

determination.

99

Figure 5.1: Arachidonic acid metabolites and inflammation. 108

Figure 5.2: The translocation of 5-lipoxygenase and cytosolic

phospholipase A2, upon cellular stimulation, to the nuclear

membrane, followed by substantial generation of

leukotrienes.

112

Figure 5.3: Cyclooxygenase and arachidonic acid metabolism. 114

Figure 5.4: COX inhibition by BE. 122

Figure 5.5: COX inhibition by LE. 123

Figure 6.1: Cholinergic neurotransmission. 132

Figure 6.2: AChE inhibition by BC. 139

Figure 6.3: AChE inhibition by galanthamine. 140

Figure 7.1: Zone of inhibition against Staphylococcus aureus (left plate)

and methicillin resistant Staphylococcus aureus (right plate)

for leaf ethanol extract at 1 mg/disc.

165

Figure 7.2: Zone of inhibition against Bacillus cereus (left plate) and

Pseudomonas aeruginosa (right plate) for leaf ethanol extract

at 1 mg/disc.

166

Figure 7.3: Time-kill plot for MSSA ATCC 11632 in presence of LE. 171

Figure 7.4: Time-kill plot for MRSA ATCC 43300 in presence of LE. 172

Figure 7.5: Time-kill plot for MSSA (clinical strain) in presence of LE. 173

Figure 7.6: Time-kill plot for MRSA (clinical strain) in presence of LE. 174

Figure 7.7: Time-kill plot for MSSA (clinical strain) in presence of BE. 175

Figure 7.8: Time-kill plot for MRSA (clinical strain) in presence of BE. 176

xv

Figure 7.9: Time-kill plot for Bacillus cereus ATCC 10876 in presence

of LE.

177

Figure 7.10: Time-kill plot for Bacillus cereus ATCC 10876 in presence

of BE.

178

Figure 7.11: Time-kill plot for Escherichia coli (clinical strain) in

presence of BE.

179

Figure 7.12: Time-kill plot for coagulase-negative Staphylococcus aureus:

O (R) (clinical strain) in presence of LE.

180

Figure 7.13: Time-kill curve for coagulase-negative Staphylococcus

aureus: O (R) (clinical strain) in presence of BH.

181


O (R) (clinical strain) in presence of BE.

182

Figure 7.15: Time-kill plot for coagulase-negative Staphylococcus.aureus:

O (S) (clinical strain) in presence of LE.

183


O (S) (clinical strain) in presence of BE.

184

Figure 7.17: Time-kill plot for Enterococcus faecalis (clinical strain) in

presence of LE.

185

Figure 7.18: Time-kill plot for Enterococcus faecalis (clinical strain) in

presence of BE.

186

Figure 7.19: Time-kill plot for Klebsiella sp. (clinical strain) in presence

of LE.

187

Figure 7.20: Time-kill plot for Klebsiella sp. (clinical strain) in presence

of BE.

188

xvi

Figure 7.21: Time-kill plot for Klebsiella pneumonia ESBL (clinical

strain) in presence of LE.

189

Figure 7.22: Time-kill plot for Klebsiella pneumonia ESBL (clinical

strain) in presence of BE.

190

Figure 8.1: Appearance of trophozoites under microscope in negative

control, positive control and with LE.

208

Figure 8.2: MTT assay of Canarium patentinervium Miq. against

Leishmania donovani.

210

Figure 9.1a: Effects of extract of leaves against MDA 468 growth. 222

Figure 9.1b: Effects of extract of barks against MDA 468 growth. 223

Figure 9.2a: Effects of extract of leaves against MCF-7 growth. 224

Figure 9.2b: Effects of extract of barks against MCF-7 growth. 225

Figure 9.3a: Effects of extract of leaves against HCT 116 growth. 226

Figure 9.3b: Effects of extract of barks against HCT 116 growth. 227

Figure 10.1: Schematic representation of the isolation and purification of

compound 1 (scopoletin), compound 2 (scoparone),

compound 3 (+-catechin), compound 4 (hyperin) and

compound 5 (cynaroside) isolated from Canarium

patentinervium Miq.

246

Figure 10.2: Schematic representation of the isolation and purification of

compound 6 (vomifoliol), compound 7 (lioxin) and

compound 8 syringic acid isolated from Canarium

patentinervium Miq.

247

Figure 10.3: The PTLC of chloroform extract of barks. 248

Figure 10.4: The chemical structure of compound 1 (scopoletin). 250

xvii

Figure 10.5: The chemical structure of compound 2 (scoparone). 251

Figure 10.6: The chemical structure of compound 3 [(+)-catechin]. 253

Figure 10.7: The chemical structure of compound 4 (hyperin). 253

Figure 10.8: The chemical structure of compound 5 (cynaroside). 255

Figure 10.9: The chemical structure of compound 6 (vomifoliol). 256

Figure 10.10: The chemical structure of compound 7 (lioxin). 257

Figure 10.11: The chemical structure of compound 8 (syringic acid). 258

Figure 11.1: Schematic of the major branch pathways of (poly) phenol

biosynthesis.

268

Figure 11.2: Main classes of phenolics. 269

Figure 11.3: 5-LOX inhibition by scopoletin and scoparone isolated from

Canarium patentinervium Miq.

284

Figure 11.4: 5-LOX inhibition by compounds isolated from Canarium

patentinervium Miq.

285

Figure 11.5: COX inhibition by (+)-catechin isolated from Canarium

patentinervium Miq.

286

Figure 11.6: AChE inhibition by isolated compounds from Canarium

patentinervium Miq.

289

Figure 11.7a: Effect of scopoletin isolated compounds from Canarium

patentinervium Miq. against growth of breast cancer cell line

MDA 468 (GI50 = 0.09µg/ml).

291

Figure 11.7b: Effect of scopoletin isolated compounds from Canarium

patentinervium Miq against growth of colon carcinoma cell

line HT-29 (GI50 = 0.17 µg/ml).

292

xviii

Figure 11.8: Time-kill plot for MSSA ATCC 11632 in presence of

scopoletin isolated from Canarium patentinervium Miq.

295

Figure 11.9: Structural features of flavonoids with high antioxidant

activity.

299

Figure 11.10: (+)-catechin and the corresponding fully oxidized ortho-

quinone product.

300

Figure A1: 1H NMR spectrum of compound 1 356

Figure A2: 13C NMR spectrum of compound 1 357











Figure A13: HMBC spectrum of compound 6 368

Figure A14: COSY spectrum of compound 6 369





Figure B1: HPLC chromatogram for compound 6 374

xix



xx

LIST OF TABLES

Page

Table 1.1: Drugs derived from plants, their clinical uses, sources and

recent plant derived drugs in global market till 2004.

4

Table 1.2: List of genera in the Burseraceae Kunth., approximate number

of species, tribal and subtribal placement, and geographic

range.

6

Table 1.3: Canarium L. species and their geographic distribution. 7

Table 1.4: Biological and pharmacological activities (in vitro) of

Canarium L. extracts and pure constituents.

14

Table 1.5: Biological and pharmacological activities (in vivo) of

Canarium L. extracts and pure constituents.

15

Table 1.6: Secondary metabolites of Canarium L. 18

Table 3.1: Preliminary phytochemical analysis of the crude extract of


54

Table 4.1: DPPH radical scavenging activity at 10 µg/ml of extract. 82

Table 4.2: Antioxidant values of Canarium patentinervium Miq. 100

Table 4.3: The mean r2 values for correlation between total phenolics

contents, total flavonoid contents and antioxidant activities

(EC50).

101

Table 5.1: Anti-inflammatory values of Canarium patentinervium Miq. 124

Table 6.1: Anti-acetylcholinesterase values of Canarium patentinervium

Miq.

138

xxi

Table 7.1: Antibacterial activity of Canarium patentinervium Miq.

(1 mg/disc) vs. ampicillin against 4 ATCC bacteria and 10

clinical bacterial strains and 3 clinical yeast strains tested by

disc diffusion assay.

167

Table 7.2: MIC values for Canarium patentinervium Miq. extracts

against 4 ATCC bacteria and 9 clinical bacterial strains and 1

clinical yeast strain.

168

Table 7.3: MBC values for Canarium patentinervium Miq. extracts

against 4 ATCC bacterias and 9 clinical bacterial strains and 1

clinical yeast strain.

169

Table 7.4: Bacteriostatic (-) and Bactericidal (+) effects of Canarium

patentinervium Miq. extracts.

170

Table 8.1: Human endoparasites in study. 199

Table 8.2: MIC values of Canarium patentinervium Miq. incubated for

24 hrs with Giardia intestinalis and Entamoeba histolytica

growing in vitro.

207

Table 8.3: Antiparasitic values of Canarium patentinervium Miq. against

Leishmania donovani (AG 83) promastigotes.

209

Table 9.1: Cytotoxic drugs developed from plant sources. 217

Table 9.2: Growth inhibition of human cancer cell lines by crude plant

extracts of Canarium patentinervium Miq.

221

Table 10.1: 1H and 13C NMR spectral data (CDCL3, 500 MHz) of

scopoletin.

250

Table 10.2: 1H and 13C NMR spectral data (CDCL3, 500 MHz) of

scoparone.

251

xxii

Table 10.3: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of (+)-

catechin.

252

Table 10.4: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of hyperin. 254

Table 10.5: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of

cynaroside.

255

Table 10.6: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of

vomifoliol.

256

Table 10.7: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of lioxin. 257

Table 10.8: 1H and 13C NMR spectral data (D2O, 500 MHz) of syringic

acid.

258

Table 11.1: Antioxidant values of isolated compounds from Canarium

patentinervium Miq.

282

Table 11.2: Anti-inflammatory values of the isolated compound from


283

Table 11.3: Anti- acetylcholinesterase values for isolated compounds from


288

Table 11.4: Growth inhibition of human cancer cell lines by isolated

compounds from Canarium patentinervium Miq.

290

Table 11.5: MIC, MBC and MBC/MIC ratio for isolated compounds from

Canarium patentinervium Miq. against Staphylococcus aureus

ATCC 11632.

294

Table 11.6: Antiparasitic values of isolated compounds from Canarium

patentinervium Miq. against Leishmania donovani (AG 83)

promastigotes and MIC values against Giardia intestinalis and

Entamoeba histolytica growing in vitro.

296

xxiii

ABBREVIATIONS

1H NMR- Proton nuclear magnetic resonance

13C NMR- Carbon nuclear magnetic resonance

5-LOX- 5-lipoxygenase

5-HPETE- 5(S)- hydroxyperoxyeicosatetraenoic acid

5-S-HETE- 5-hydroxy- 6,8,11,1-eicosatetraenoic acid

µg- Microgram

µl- Microliter

µM- Micromolar

oC- Celcius

AAE- Ascorbic acid equivalent

AChE- Acetylcholinesterase

ACh- Acetylcholine

AD- $O]KHLPHU¶VGLVHDVH

AIDS- Acquired immunodeficiency syndrome

amu- Atomic mass unit

AST- Antimicrobial susceptibility testing

ATCC- American Type Culture Collection

BChE- Butyrylcholinesterase

BDE- Bond dissociation energy

CAT- Choline acetyltransferase

CC- Column chromatrography

CCl4- Carbon tetrachloride

xxiv

cfu- Colony forming units

cm- Centimeter

CNS- central nervous system

COSY- correlation spectroscopy

COX-1- Cyclooxygenase-1



cPLA2- Cytosolic phospholipase A2

d- Doublet

dd- Doublet of doublet

D-GaIN- D-galactosamine

DMAPP- Dimethyl allyl diphosphate

DMSO- Dimethyl sulfoxide

DPPH- 1,1,-diphenyl-2-2-picrylhydrazyl

DTNB- 5,5´-Dithio-bis(2-nitrobenzoic) acid

EC50- Effective concentration at 50 % activity

EDTA- Ethylene diamine tetra-acetic acid

FC- Folin-Ciocalteu

FLAP- 5-lipoxygenase activating protein

FRAP - Ferric reducing antioxidant power

g- Gram

GAE- Gallic acid equivalent

GC- Gas chromatography

GOT- Glutamic oxaloacetic transaminase

GPT- Glutamic pyruvic transaminase

xxv

HAT- Hydrogen atom transfer reaction

HMBC- heteronuclear multiple-bond correlation spectroscopy

HPLC- High performance liquid chromatography

hr- Hour

HO2- Hydroperoxyl

IC50- Inhibitory concentration at 50 % activity

iNOS- Inducible NO (nitric oxide) synthase

INT- p-iodonitrotetrazolium

IP- Ionization potential

IPP- Isopentyl diphosphate

IR- Infrared spectroscopy

i.p- Intra peritoneal

kg- Kilogram

L- Litre

lb- Pound

LOO- Peroxy radical

LTA4- Leukotriene A4

LTB4- Leukotriene B4

M- Molar

m- Multiplet

mM- Milimolar

MBC- Minimum bactericidal assay

mg- Miligram

MHB- Mueller Hilton broth

MHA- Mueller Hilton agar

xxvi

ml- Mililitre

MIC- Minimum inhibitory concentration

Min- Minutes

MS- Mass spectroscopy

MTT- Methylthiazol tetrazolium

NCE- New chemical entities

NDGA- Nordihydroguairetic acid

NCCLS- National Committee for Clinical Laboratory Standards

nm- Nanometer

NMR- Nuclear magnetic resonance

NSAID- Non-steroidal anti-inflammatory drugs

NO2- Nitrogen dioxide

N2O3- Dinitrogen trioxide

O2- - Superoxide

ONOO- Peroxy nitrate

ORAC- Oxygen radical absorbance capacity

PC- Phospholipase C

PGs- Prostaglandins

PGG2- Prostaglandin G2

PGI2- Prostacyclin

PLA2- Phospholipase A2

PNS- Peripheral nervous system

ppm- Parts per million

PTLC- Preparative thin layer chromatography

RA- Rheumatoid arthritis

xxvii

ROS- Reactive oxygen species

RNS- Reactive nitrogen species

Rt- Retention time

ROO- Peroxyl

s- Singlet

SET- Single electron transfer

SD- Standard deviation

SOD- Superoxide dismutase

t- Triplet

TEAC- Trolox equivalent antioxidant capacity

TLC- Thin layer chromatography

TMPD- N,N,N¶,N¶-tetramethyl-p-phenylenediamine

TPTZ- 2,4,6-tripyridyl-s-triazine

TSA- Tryptic soya agar

TSB- Tryptic soya broth

TxA2- Thromboxane A2

UV- Ultra-violet spectroscopy

WST-1- (2-(4-Iodophenyl)- 3-(4- nitrophenyl)-5-(2,4-disulfophenyl)- 2H-

tetrazolium, monosodium salt)

XO- Xanthine oxidase

xxviii

TABLE OF CONTENTS

Page Declaration

ii

Dedication

iii

Acknowledgements

iv

Abstract

v

Publications arising from this study

x

Conference proceedings and talks related to this study

xi

List of figures

xii

List of tables

xx

Abbreviations

xxiii

Table of contents

xxviii

CHAPTER 1: GENERAL INTRODUCTION

1

1.1 Introduction 1

1.2 An ethnopharmacological research 2

1.3 An introduction to the family Burseraceae Kunth. and genus

Canarium L.

3

1.3.1 The family Burseraceae Kunth 3

1.3.2 The genus Canarium L. 5

1.3.3 Botanical features of Canarium L. 8

1.4 Traditional and medicinal uses of Canarium L. 9

xxix

1.4.1 In vitro and in vivo pharmacological activities of Canarium L. 10

1.5 The phytochemistry of Canarium L. 13

1.6 The commercial uses of Canarium L. produces 17

1.7 Selection of plant material

42

1.7.1 The selection of Canarium patentinervium Miq.

43

1.7.2 The selection of bioassays performed

43

1.8 Aims of study

44

1.9 Objectives of study

44

CHAPTER 2: PLANT COLLECTION AND EXTRACTION

47

2.1 Brief introduction to Canarium patentinervium Miq. 47

2.2 Plant collection and extraction 47

CHAPTER 3: PHYTOCHEMICAL METHODS

50

3.1 Introduction

50

3.2 Phytochemical assay protocol

50

3.3 Results

52

3.4 Discussion

53

3.5 Conclusion

53

CHAPTER 4: THE ANTIOXIDANT ACTIVITY

59

4.1 Introduction 59

xxx

4.1.1 Free radicals and their mechanism of action

59

4.1.2 Natural antioxidants

61

4.1.3 Therapeutic potential of phenolic substances

62

4.1.4 Antioxidant potential of Canarium L.

63

4.2 Materials and methods

64

4.2.1 Antioxidant determination assays

64

4.2.2 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay 66

4.2.2.1 Principle of the assay

66

4.2.2.2 Colorimetric spectrophotometric assay

68

4.2.3 ¶-Azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid)

(ABTS) assay

69


69


71

4.2.4 Ferric reducing ability of plasma (FRAP) assay

72

4.2.4.1 Principle of assay 72

4.2.4.2 Colorimetric spectrophotometric assay 74

4.2.5 ȕ-carotene bleaching assay 75



4.2.6 Superoxide dismutase assay 77



4.2.7 Total phenolic content determination

78


xxxi


4.2.8 Total flavonoid content determination 79

4.2.8.1 Principle of assay

79


4.2.9 Statistical analysis 79

4.3 Results 80

4.3.1 DPPH assay 81

4.3.2 ABTS assay 81

4.3.3 FRAP assay 81

4.3.4 ȕ-carotene bleaching assay 85

4.3.5 Superoxide dismutase assay 85

4.3.6 Total phenolic content determination 85

4.3.7 Total flavonoid content determination 86

4.3.8 The antioxidant values, total phenolic and flavonoid content

and their correlations

86

4.4 Discussion 102

4.5 Conclusion 105

CHAPTER 5: THE ANTI-INFLAMMATORY ACTIIVTY 106

5.1 Introduction 106

5.1.1 Inflammation and arachidonic system 106

5.1.2 The lipoxygenase system 110

5.1.3 The cyclooxygenase system 111

5.1.4 Dual inhibitors 115

xxxii

5.1.5 Anti-inflammatory potential of Canarium L. 115

5.2 Materials and methods 116

5.2.1 Anti-inflammatory determination assays 116

5.2.2 The 5-LOX inhibition assay 117


5.2.2.2 Protocol 118

5.2.3 The peroxidase endpoint assay for COX-1 and COX-2 118




5.3 Results 120

5.3.1 The 5-LOX inhibition assay 120

5.3.2 The peroxidase endpoint assay for COX-1 and COX-2 121

5.4 Discussion 121

5.5 Conclusion 127

CHAPTER 6: ANTI-ACETYLCHOLINESTERASE ACTIVITY 128


6.1.1 3DWKRSK\VLRORJ\RI$O]KHLPHU¶VGLVHDVH 128

6.1.2 Acetylcholine and its functions 130

6.1.3 Distribution and function of cholinesterase 130

6.1.4 Cholinesterase inhibitors 131

6.1.5 Plants as potential anticholinesterase inhibitors 134

xxxiii


6.2.1 Anti-acetylcholinesterase determination assays 135




6.3 Results 137

6.4 Discussion 141

6.5 Conclusion 142

CHAPTER 7: ANTIMICROBIAL ACTIVITY 144


7.1.1 Infectious disease 144

7.1.2 Chemotherapeutic agents: Factors affecting their effectiveness 144

7.1.3 Drug resistance 145

7.1.4 Natural products and their role in drug discovery 146

7.1.5 Canarium species and their known antimicrobial activity 147


7.2.1 Disc diffusion assay 148

7.2.1.1 Principle of method 148


7.2.2 Minimum inhibitory concentration assay 153



7.2.3 Minimum bactericidal concentration (MBC) 157

xxxiv



7.2.4 Time-kill assay 157




7.3 Results 159


7.3.2 MIC assay 160

7.3.3 MBC assay and MBC/MIC ratio 161


7.4 Discussion 163


7.4.2 MIC, MBC and MIC/MBC ratio 191


7.5 Conclusion 196

CHAPTER 8: ANTIPARASITIC ACTIVITY 198


8.1.1 Parasitic infection: causative factor and treatment 198

8.1.2 Natural products and parasitic defence 201


8.2.1 Principle of method 202

8.2.2 Protocol 201

xxxv

8.2.2.1 Antiparasitic assay against Giardia intestinalis

and Entamoeba histolytica

201

8.2.2.1.1 Test organisms 202

8.2.2.1.2 Cultivation of organisms 203

8.2.2.1.3 Antiprotozoal assay 203

8.2.2.2 Antiparasitic activity against Leishmania

donovani promastigotes

204


8.3 Results 205

8.4 Discussion 206

8.5 Conclusion 212

CHAPTER 9: ANTICANCER ACTIVITY 213


9.1.1 Carcinogenesis 213

9.1.2 Natural products and carcinogenesis defence 214


9.2.1 Principle of method 218

9.2.2 Protocol 218

9.2.2.1 Cell lines and cell culture 218

9.2.2.2 Preparation of plant samples 219

9.2.2.3 The MTT assay 219


xxxvi

9.3 Results 220

9.4 Discussion 220

9.5 Conclusion 228

CHAPTER 10: ISOLATION AND IDENTIFICATION OF

COMPOUNDS

229


10.1.1 Secondary metabolites 229

10.1.2 Isolation methods 232

10.1.3 Crude fractionation 233

10.1.4 Chromatography 233

10.1.4.1 Thin layer chromatography (TLC) 234

10.1.4.2 Liquid chromatography (LC) 236

10.1.4.3 Size exclusion chromatography 237

10.1.4.4 High performance liquid chromatography system 239

10.1.5 Structure elucidation 240

10.1.5.1 Nuclear magnetic resonance (NMR) 241


10.2.1 Isolation of compound 1-5 (crude fractionation) 242

10.2.2 Isolation of compound 1 & 2 242

10.2.2.1 Silica gel column chromatography 242

10.2.2.2 Preparative thin layer chromatography (PTLC) 243

10.2.3 Isolation of compound 3,4 & 5 243

10.2.3.1 Size-exclusion column chromatography 243

xxxvii

10.2.4 Isolation of compound 6-8 244

10.2.4.1 Isolation of compound 6-8 (PTLC and solvent

partioning

244

10.2.4.2 Isolation of compound 6-8 (HPLC) 245

10.2.5 Nuclear magnetic resonance (NMR) 249

10.3 Results 250

10.4 Discussion 259

10.5 Conclusion 263

CHAPTER 11: BIOACTIVITY OF ISOLATED SECONDARY

METABOLITES

264


11.1.1 Terpenes and phenols 264

11.1.2 General antioxidant mechanism of phenolics 266

11.1.3 Flavonoids 270

11.1.4 Phenolic acids 272

11.1.5 Coumarins 273


11.2.1 Antioxidant capacity assays 275


11.2.3 Anti-acetylcholinesterase determination assay 276

11.2.4 Anticancer assay 277

11.2.5 Antibacterial assays 277

11.2.6 Antiparasitic assays 278

xxxviii

11.2.7 Formulation of scopoletin 278


11.3 Results 279

11.3.1 Antioxidant capacity assays 279


11.3.3 Anti-acetylcholinesterase detmination assay 281

11.3.4 Anticancer assay 287

11.3.5 Antibacterial assays 287

11.3.6 Antiparasitic assays 293

11.4 Discussions 293

11.4.1 Antioxidant activity 298

11.4.2 Anti-inflammatory activity 303

11.4.3 Anti-acetylcholinesterase activity 305

11.4.4 Antiparasitic activity 306

11.4.5 Anticancer activity 307

11.4.6 Antibacterial activity 308

11.4.7 Formulation of scopoletin as a gel 308

11.5 Conclusion 310

CHAPTER 12: GENERAL CONCLUSIONS 312

CHAPTER 13: RECOMMENDATIONS FOR FUTURE WORK 321

REFERENCES 324

APPENDIX A: NMR DATA 356

xxxix

APPENDIX B: HPLC CHROMATOGRAMS 374

APPENDIX C: PUBLICATIONS 377

APPENDIX D: CONTINUOUS EDUCATION 383

1

CHAPTER 1

GENERAL INTRODUCTION

1.1 Introduction

Pharmacognosy is not a familiar term, even to many scientists which is defined as the

study of crude drugs of plant and animal origin. The scope of pharmacognosy is also

defined as the study of physical, chemical, biochemical and biological properties of

drugs, drug substances, or potential drugs or drug substances of natural origin as well

as the search for new drugs from natural origin as well as the search for new drugs

from natural sources [1]. Natural products offer unmatched chemical diversity with

structural complexity and biological potency. It is estimated about 100-fold higher hit

rate for natural products over synthetic compounds. Natural products occupy different

chemical space that is sometimes difficult to access compared with synthetic

compounds. Not only do the natural product databases contain many more scaffolds,

but an important proportion of the ring systems are not found at all in other drug

databases. Such unexploited scaffolds represent promising new starting points in drug

discovery.

Natural products compounds not only serve as drugs or templates for drugs but lead to

a better understanding of targets and pathways involved in disease process. They also

create opportunities for additional drug targets to be identified and exploited in these

pathways. The elucidation of the anti-inflammatory mechanism of action of aspirin

led to the discovery of the cyclooxygenase isozymes COX-1 and COX-2, which were

used in the development of novel anti-inflammatory drugs [2]. Over half of the

2

ZRUOG¶VWRSEHVW-selling pharmaceuticals drugs in 1991 owed their origin to natural

products. Higher plant-derived products represent around 25% of the total number of

clinically used drugs and include the classical drugs atropine, codeine, digoxin,

morphine and quinine [1].

Number of drug molecules obtained and developed industrially from plants and used

in modern medicines increased drastically from 121 in 1995, 130 in 1997, 143 in 2000

and 166 in 2006. From 1981 and 2000, 61% of the small molecule- new chemical

entities (NCE) that were introduced as drugs worldwide can be traced to or were

inspired by natural products. Between 2001 and 2005, 23 new drugs derived from

natural products were introduced for the treatment of bacterial and fungal infection,

FDQFHU GLDEHWHV G\VOLSLGHPLD DWRSLF GHUPDWLWLV $O]KHLPHU¶V GLVHDVH DQG JHQHWLF

disease such as tyrosinaemia and Gaucher disease [3]. Table 1.1 lists the drugs

derived from plants, their clinical uses and sources as well as recent plant-derived

drugs in the global market.

1.2 An ethnopharmacological research

The study of plants used in traditional medicine requires the effective integration of

information on chemical composition of extracts, pharmacological activities of

isolated compounds, as well as indigenous knowledge of traditional healers. The

acquisition of ethnobotanical information remains an empirical aspect in any such

study [4]. There is a great need and ethical obligation to accurately document

investigative findings on plants used for health purposes. This will also aid in the

3

efficient preservation and conservation of traditional knowledge which may

potentially benefit society in general.

1.3 An introduction to the family Burseraceae Kunth. and genus Canarium L.

1.3.1 The family Burseraceae Kunth.

The Burseraceae Kunth is one of the nine flowering plant families belonging to the

order Sapindales Juss. ex Bercht. & J. Pearl that comprise the monophyletic group

(5700 species), whose first known fossils appear in Europe 65 million years ago (Ma).

The Burseraceae Kunth. consists of approximately 18 genera and 700 species. This

family is divided into 3 tribes namely Canariceae, Protieae which are determined to be

monophyletic and Burserae which was shown to be polyphyletic. The list of genera in

the Burseraceae Kunth., approximate number of species, tribal and subtribal in the

Burseraceae Kunth., approximate number of species, tribal and subtribal placement,

and geographic range of genera are shown in Table 1.2. The family is distributed

pantropically across a broad range of low-elevation, frost-free habitats including

rainforest, dry deciduous forest and deserts [5]. The Burseraceae Kunth. are best

known for producing resins of economic, medicinal, and cultural values such as

frankincense, myrrh and copal [6].

4

Table 1.1 : Drugs derived from plants, their clinical uses, sources and recent plant derived drugs in global market till 2004 [3][7].

Drug Clinical Use/Action Plant source

Atropine Anticholinergic Atropa belladonna Linn.

Colchicine Antitumor, antigout agent Colchicum autumnale L.

Digitoxin Cardiotonic Digitalis purpurea L.

Emetine Emetic, amoebicide Cephaelis ipecacuanha (Brot.) A. Rich.

Morphine Analgesic Papaver somniferum L.

Pilocarpine Parasympathomimetic Pilocarpus jaborandi (Holmes.)

Quinine Antimalarial Cinchona ledgeriana (Howard) Bern.

Apomorphine HCL Potent dopamine receptor agonist Papaver somniferum L.

3DUNLQVRQ¶VGLVHDVH

Tiotropium bromide Longer-acting antibronchospasm Atropa belladonna Linn.

Nitisinone For hereditary tyrosinaemia type-1 Callistemone citrinus (Curtis) Skeels.

Galanthamine HBr Selective acetylcholinesterase Galanthus nivalis L.

,QKLELWRU$O]KHLPHU¶VGLVHDVH

Arteether Antimalarial Artemisia annua L.

5

1.3.2 The genus Canarium L.

The genus Canarium L. probably originated from the North American continent, not

Gondwanaland [5]. This clade embraces 75 species of trees which are mainly found in

tropical Asia and the Pacific, and a few species in tropical Africa [8], about 9 species

were found in the Philippines [8]. The geographical centre of their genetic diversity is

the Molucca Islands of eastern Indonesia, but their centre of cultivated diversity is

undoubtedly western Melanesia. Twenty to 25 species are found in the South Pacific,

of which 21 are in Papua New Guinea [9], eight in the Solomon Islands [10] and 3 or

4 in Vanuatu [11]. The known Canarium species and their distribution are listed in

Table 1.3.

The word Canarium L. GHULYHV IURP WKH 0DOD\ QDPH µNDQDUL¶[8]. Canarium L.

species often produce edible kernels, called canarium nut of commercial interest:

Canarium indicum L., Canarium solomonense B.L.Burtt, Canarium harveyi Seem,

Canarium odontophyllum Miq. and Canarium album L. Another economical interest

of Canarium L. species, is the production of resins used in foods (Canarium

luzonicum Miq.), in the making of incense and varnishing. In spite of these

commercial potentials of Canarium L., little attention has been given to the collection

and conservation of Canarium L. species [12]. The genetic diversity thus derogates at

an alarming rate.

6

Table 1.2: List of genera in the Burseraceae Kunth., approximate number of species, tribal

and subtribal placement, and geographic range [5].

Bursereae Burserinae

Bursera Jacq. ca. 100 spp. Caribbean, Mexico, Central, and S. America Commiphora Jacq. ca. 190 spp. Africa, India, S. America

Boswelliinae Aucoumea Pierre 1 sp. W. Africa Beiselia Forman 1sp. SW Mexico Boswellia Roxb. ca. 30 spp. NE Africa, Arabia, India Triomma Hook. f. 1sp. W Malesian region Unnamed subtribe Garuga Roxb. 4 spp. India, SE Asia

Canarieae Canarium L. ca. 105 spp. SE Asia, Malaysia, Africa Dacryodes Vahl 66 spp. Caribbean, Mexico, C. and S. America, SE Asia, Africa Haplolobus H.J. Lam 22 spp. E. Malaysia Pseudodacryodes R. Pierlot 1 sp. Central Africa Rosselia Forman 1 sp. Rossel Island, New Guinea Santiria Blume 24 spp. W. Malesian region, Philipines, Moluccas, New Guinea, Africa Scutinanthe Thwaites 2 spp. Sri Lanka, S. Myanmar, Celebes, Sumatra, Malay Peninsula,Borneo Trattinnickia Willd. 13 spp. C. and S. America

Protieae Crepidospermum Hook. f. 6 spp. S. America Protium Burm. f. 150 spp. Mexico, C. and S. America, Africa, SE Asia Tetragastris Gaertn. 9 spp. Central and S. America

Garuga was placed informally within the Bursereae by Harley and Daly (1995).

7

Table 1.3: Canarium L. species and their geographic distribution [13]. Species Distribution

C. acutifolium (DC.) Merr. Moluccas, New Guinea, Central Celebes C. album (Lour.) Raeusch. Annam, Tonkin, S. China, Hainan C. apertum H.J. Lam Sumatra, Malay Peninsula, Borneo C. asperum Benth. Solomon Is., Bawean and Kangean Is subsp. asperum Lesser Sunda Is., Borneo, Philippines, subsp. papuanum (H.J. Lam) Leenh. Celebes, Moluccas, New Guinea C. australianum F.v.M N. Australia, SE New Guinea C. baileyanum Leenh. Queensland, NS Wales C. balansae Engl. Loyalty Is. C. balsamiferum Willd. Celebes, Moluccas, New Guinea C. luzonicum (Bl.) A. Gray Philippines C. macadamii Leenh. New Guinea C. madagascariense Engl. Madagascar, Tanganyika, subsp. Madagascariense Mozambique subsp. Obtusifolium (S.Elliot) Leenh. C. maluense Laut. Central Celebes, Moluccas, New Guinea subsp. maluense subsp. borneense Leenh. C. manii King Middle and S. Andaman C. megacarpum Leenh. W. New Guinea C. megalanthum Merr. Sumatra, Malay Peninsula, Borneo C. merrillii H. J. Lam Borneo C. muelleri F.M. Bailey Australia (Queensland) C. odontophyllum Miq. Sumatra, Borneo, Philippines C. oleiferum Baill. New Caledonia C. oleosum (Lamk) Engl. New Britain, Lesser Sunda Island.,

N. Celebes, Moluccas, New Guinea C. ovatum Engl. Philippines C. paniculatum (Lamk) Benth. ex Engl. Mauritius C. parvum Leenh. Tonkin, N. Annam C. patentinervium Miq. Sumatra, Malay Peninsula, Banka,

Anambas Island., Borneo C. perlisanum Leenh. Malay Peninsula C. piloso-sylvestre Leenh. W. New Guinea C. pilosum Benn. Sumatra, Malay Peninsula, Borneo subsp. pilosum subsp. borneensis Leenh. C. pimela Leenh. S. China, Hainan, Tonkin, Laos,

Annam, Cambodia C. polyphyllum K. Sch. New Guinea C. pseudodecumanum Hochr. Sumatra, Malay Peninsula, Borneo C. pseudopatentinervium S. Sumatra, Banka, Borneo H.J. Lam C. pseudosumatranum Leenh. Malay Peninsula C. rigidum (Bl.) Zipp. ex Miq. New Guinea C. salomonense B.L. Burtt Solomon Island., E. New Guinea subsp. salomonense subsp. papuanum C. samoense Engl. Samoa C. schlecteri Laut. New Britain, E. New Guinea

8

Table 1.3: Canarium L. species and their geographic distribution [13]. (continuation) Species Distribution

C. schweinfurthii Engl. Trop. W and Central Africa C. smithii Leenh. Fiji C. strictum Roxb. SW Deccan, Sikkim, Assam, Upper Burm C. subulatum Guill. S. China, Hainan, Tonkin, Laos, Annam, Cambodia C. sumatranum Boerl. Sumatra, Malay Peninsula and Koord. C. sylvestre Gaertn. Moluccas, New Guinea C. trifoliatum Engl. New Caledonia C. trigonum H.J. Lam Central Celebes C. vanikoroense Leenh. New Hebrides, Fiji C. vitiense A. Gray Fiji C. vrieseanum Engl. Philippines, Central and N. Celebes C. vulgare Leenh. Kangean and Bawean Island., Lesser Sunda Island., Celebes, Moluccas C. whitei Guill. New Caledonia C. zeylanicum (Retz.) Bl. Sri Lanka

1.3.3. Botanical features of Canarium L.

The members of the genus Canarium L. consist of medium to large buttressed trees

up to 40-50m tall, or rarely a shrub. The barks are greenish grey, fawn or light yellow

brown that are usually smooth, scaly or dippled with many small lenticels. Outer bark

are thin while the inner barks are pinkish brown or reddish brown, laminated, soft and

aromatic with a clear sticky or rarely oily exudate. The stems are usually terete. The

leaves are pinnate, spiral and stipulated. The rachis is terete flattened to channeled

swollen at base, and bears 5-21 folioles. The folioles are oblique at base, entire,

dentate or serrate at margin, often thick and acuminiate at apex. The secondary nerves

are arching and joined near margin. The tertiary nerves are reticulate. The

infloresence is an axillary or terminal panicle. The calyx is cupular. The corolla

includes 3 creamy petals. The androecium comprises a whorl of 6 stamens. The disc

within the stamens is 6 lobed. The gynaecium consist of 3 carpels united into a 3

9

lobular ovary. The drupes are seated on a persistent enlarged calyx and enclose a

woody stone [14].

1.4 Traditional and medicinal uses of Canarium L.

Elemi (British Pharmaceutical Codex, 1934) is an oleoresin exuded through the bark

of Canarium luzonicum Miq. or Canarium commune L. which has been used in the

form of an ointment as a stomach stimulant and as an expectorant [15]. The barks of

Canarium indicum L. has been used for chest pains where else the oil has been

patented for treatment of arthritis pain and the oleoresin of the tree is applied as a

poultice for ulcerated wounds. The resin of Canarium tonkinense Engl. has been used

as a stimulant, rubefacient and anti-rheumatic when applied externally. The oleoresin

has been applied as ointment for ulcers [16]. The dried fruit of chinese olive or

Canarium album (Lour.) Raeusch. is used in China and used to treat bacterial and

viral infections, inflammation, poisoning and for detoxification [17].

In Chinese folk medicine, the dried fruits of Canarium album (Lour.) Raeusch have

been used for treatment of angina, dysentery, snake bites, cough-hematemesis,

enteritis, diarrhoea, toxicosis from swellfish and alcohol [18]. Canarium

schweinfurthii Engl. is used by traditional healers as a remedy for diabetes mellitus in

southern Senegal [19] while in Congo and Central African Republic the plant is used

in fever, as stimulant, emollient, in post-partum pain, constipation, malaria, diarrhoea,

sexual infections and rheumatism [20]. In Indonesia, the bark of Canarium littorale

Bl. is used to make a decoction taken to heal haemorrhoids [21].

10

1.4.1 In vitro and in vivo pharmacological activities of Canarium L.

Only 12 % of 75 species have been studied for their pharmacological activities.

Extracts and pure compounds derived from Canarium L. were reported to have a

variety of pharmacological activities of which antioxidant, antibacterial, antifungal,

antitumor, anti-inflammatory, hepatoprotective, analgesic and anti-diabetic (Table

1.4).

i. Antioxidant

Antioxidant activities were reported in Canarium album (Lour.) Raeusch, Canarium

odontophyllum Miq. and Canarium schweinfurthii Engl. A tonic soup made of

Canarium album (Lour.) Raeusch used mainly in China displayed significant

antioxidant activity by 1,1,-diphenyl-2-2-picrylhydrazyl (DPPH) and ferric reducing

antioxidant power (FRAP) assay [22]. Tannins extracted from the leaves, twigs and

stem barks of Canarium album (Lour.) Raeusch showed potent antioxidant activity in

the DPPH radical scavenging activity with IC50 values of 56.86 µg/ml, 62.31 µg/ml

and 54.80 µg/ml respectively and ferric reducing power of 4.28, 3.74 and 4.49 mmol

AAE/g equivalent of dried tannin [23]. Pure compounds from this species includes

brevifolin, hyperin and ellagic acid which showed free radical scavenging activity in

DPPH assay [24].

The essential oil of Canarium schweinfurthii Engl. was tested for the antioxidant

DFWLYLW\ZLWK WKH'33+DVVD\DQGE\ȕ-carotene bleaching test. It exhibited highest

antioxidant activity at 150 µg/ml activity in both assay [25]. The ethyl acetate fraction

11

of peel of Canarium odontophyllum Miq. exhibited 95±1.00% scavenging activity at

the concentration of 40 µg/ml [26][27].

ii. Antibacterial and antifungal activities

Antibacterial activities were reported in Canarium schweinfurthii Engl. and Canarium

patentinervium Miq. Dichloromethane extract of Canarium schweinfurthii Engl. had

bactericidal activity against Gram-negative Vibrio cholerae with minimum inhibitory

concentration (MIC) of 0.62 mg/ml while the ethylacetate extract was active against

Gram-positive and Gram-negative bacteria namely Staphylococcus aureus and

Proteus vulgaris with MIC values of 10 mg/ml and 5 mg/ml respectively. Ethanol

extract was active against Gram-negative Vibrio cholerae and Proteus vulgaris with

MIC values of 0.62 mg/ml and 10 mg/ml respectively [28]. In a separate disc

diffusion assay, the essential oil of Canarium schweinfurthii Engl. abrogated the

survival of Gram-negative Salmonella enterica, Gram-positive Streptococcus pyogens

and Staphylococcus aureus with an inhibition zone of 27 mm, 25 mm and 18 mm

respectively. However the author did not inform on the concentration of the extract

per disc. The oil was also fungicidal against Candida albicans with an inhibition

zone of 23 mm [25].

iii. Hepatoprotective activities

Hepatoprotective activity was exhibited in Canarium manii King. and Canarium

album (Lour.) Raeusch. The biflavanoid agathisflavone from Canarium manii King.

preserved the integrity of the liver cells membrane of rodents as evidenced by the

12

decrease in the CCl4-induced rise of glutamic oxaloacetic transaminase (GOT) and

glutamic pyruvic transaminase (GPT) levels. GPT which is predominantly found in

the liver showed a dose-dependent and significant reduction [29]. The triterpenes urs-

12-ene-Įȕ-diol, olean-12-ene-Įȕ-diol and urs-12-ene-ȕȕ-diol from from

Canarium album (Lour.) Raeusch markedly reduced the amount of alanine

aminotransferase leakage from the primary cultured hepatocytes intoxicated with 0.2

mM of D-galactosamine (D-GaIN) [30]. The phenols brevifolin and ellagic acid

protected rat hepatocytes against D-GaIN-induced insults [24].

iv. Other Biological Activities

The essential oil of Canarium schweinfurthii Engl. collected from the region of

Cameroon inhibited the enzymatic activity of lipoxygenase with an IC50 value of 62.6

ppm [31]. However in a separate study of the same species collected from central

African region did not show any activity in the cotton pellet induced granuloma

formation experiment [20]. This may suggest possible evidence of variety between

secondary metabolites constituents according to regions. The essential oil of

Canarium schweinfurthii Engl. at a dose of 1, 2 and 3 mg/kg i.p. displayed potent

analgesic effect in the acetic acid-induced writhing and hot plate experiments [20].

Anti-diabetic activity was reported in the methanol/methylene chloride extract of stem

bark of Canarium schweinfurthii Engl. At a dose of 300 mg/kg there was 67.1 %

reduction in blood glucose levels after a once daily subcutaneous injection on

streptozotocin-induced diabetic male rats over 14 days, versus insulin that had 76.8 %

reduction. Weight gain was only 6.6 % as opposed to untreated rats that had lost 14.1

13

% of body weight. There was also significant reduction in food and fluid consumption

by 68.5 % and 79.7 %. These results showed the extract could reverse hyperglycemia,

polyphagia and polydipsia provoked by streptozotocin, thus having anti-diabetic

activity [19].

1.5 The phytochemistry of Canarium L.

Isolation and structure elucidations of secondary metabolites in Canarium L. has been

carried out since the 50¶s [32]. Majority of investigations include the resin and the

fruit of the species. To date about 96 compounds have been isolated from 9 species,

which are Canarium schweinfurthii Engl., Canarium boivinii Engl., Canarium

odontophllum Miq., Canarium manii King., Canarium album (Lour) Raeusch,

Canarium zeylanicum (Retz.) Blume, Canarium commune L., Canarium muelleri

F.M. Bailey and Canarium bengalense Roxb. The extensively researched species are

Canarium schweinfurthii Engl. and Canarium album (Lour.) Raeusch

[17][18][23][33][34].

14

Table 1.4: Biological and pharmacological activities (in vitro) of Canarium L. extracts and pure

constituents

Extract/Compound Species Pharmacological activity References

Polymeric

procyanidins(tannins)

from leaves, twigs and

stem bark

Canarium album

(Lour.) Raeusch

Significant DPPH radical

scavenging activity,

Ferric reducing antioxidant

activity

[23]

Ethyl acetate fraction

of the fruit peel

Canarium

odontophyllum

Miq.

Antioxidant activity with

DPPH assay, FRAP assay and

hemoglobin oxidation assay

[26]

Carotenoids from

peel,pulp and seed

extracts

Canarium

odontophyllum

Miq.

Significant antioxidant activity

with beta-carotene bleaching

assay, ABTS assay, DPPH

assay and hemoglobin

oxidation assay

[27]

Essential oil of resins Canarium

schweinfurthii Engl.

Bactericidal for Enterococcus

faecalis, Listeria innocua,

Salmonella enterica,

Staphylococcus aureus,

Staphylococcus camorum

Fungicidal for Candida

albicans

$QWLR[LGDQWIRU'33+DQGȕ-

carotene bleaching test

[25]

Essential oil of

resins(monoterpenes

hydrocarbon)

Canarium


Significant anti-inflammatory

activity via lipooxygenase

method with IC50 of 62.6 ppm

[31]

Brevifolin, ellagic

acid and hyperin

Canarium album

(Lour.) Raeusch

Significant antioxidant activity

and inhibitory effect on lipid

peroxidation assay

[24]

Dichloromethane

extract of barks

Canarium


Antimicrobial activity against

V.cholerae

[28]

Extract of whole plant Canarium album

(Lour.) Raeusch

Antioxidant activities in DPPH

and FRAP assay

[22]

15

Table 1.5: Biological and pharmacological activities (in vivo) of Canarium L. extracts and pure

constituents

Extract/Compound Species Pharmacological activity References

Agathisflavone

(biflavanoid)

Canarium manii

King.

Hepatoprotective activity

against experimentally-induced

carbon tetrachloride-

hepatotoxicity in rats and mice

[29]

urs-12-ene-Įȕ-

diol, olean-12-

ene-ĮȕGLRO(triterpene)

Canarium album

(Lour.) Raeusch

Hepatoprotective activity in

primary cultured rat

hepatocytes intoxicated with D-

galactosamine

[30]

brevifolin, ellagic acid Canarium album

(Lour.) Raeusch

Reduction of carbon

tetrachloride induced liver

damage in mice. Reduction in

elevated GPT and GOT levels

after intraperitoneal

administration

[24]

Essential oil of

resins(composed

mainly of nerolidol

and octylacetate)

Canarium


Significant analgesic effect

using acetic acid-induced

writhing and hot plate methods

with swiss mice

[20]

Methanol/methylene

chloride extract of

stem barks

Canarium


Anti-diabetic activity that

reverses hyperglycemia,

polyphagia and polydipsia in

streptozotocin-induced diabetic

rats. Significant reduction 69.9

% reduction in blood glucose

level after 14 days at 300

mg/kg

[19]

16

The isolation and separation technique is very much dependent on the type of

fractions. Essential oils are analysed with gas chromatography (GC) and mass

spectroscopy (MS) [20][31][35]. Other substances are separated with liquid

chromatography using different solvent mixtures with silica gel [29][36], charcoal

[37], sephadex [18] and multiple column packing such as AB-8 adsorption resin,

polyamide, and TSK Toyopearl HW-40(S). Other types of analytical techniques

include thin layer chromatography (TLC) and high performance liquid

chromatography (HPLC) [17][27][34][38][39].

The structures are mainly established by mass spectroscopy (MS), ultra-violet

spectroscopy (UV), infrared spectroscopy (IR) and 1H and/or 13C nuclear magnetic

resonance (NMR). 1H and/or 13C spectroscopy is probably the most useful method in

structure elucidation [29]. Among the secondary metabolites isolated from members

of the genus Canarium L. are terpenes (monoterpenes, triterpenes, tetraterpenes like

carotenoids, sesquiterpenes, cyclohexane and sterols), carboxylic acids, coumarins,

furans, lipids and phenols (flavonoids, tannins, phenolic acids). The main secondary

.metabolites isolated so far from the genus Canarium L. consists of terpenes with 58

compounds and flavonoids with 11 compounds. The profile of all known secondary

metabolites of Canarium L. as found in literature are included in Table 1.6 and their

structures are included in Figure 1.1.

17

1.6 The commercial uses of Canarium L. produces

(OHPL GHULYHG IURP WKH $UDELF µ$O-ODPL¶ LV D FROOHFWLYH WHUP DSSOLHG WR VHYHUDO

oleoresins obtained from different plants of the family Burseraceae Kunth. The most

important and widely known of these oleoresins is Manila Gum Elemi which exudates

from the trunk of Canarium luzonicum Miq. or Canarium commune L. The natural

constituent of elemi oil is elemicin. Variety of foodstuffs are flavoured with elemi oil

and in Europe its used in spices and seasonings. In US elemi oil is also used in

fragrances to approximately 1000 lb/year [40]. At present, at least 4 species of

Canarium L. nuts are of economic importance. Canarium ovatum Engl. (known

ORFDOO\ DV µSLOL¶ DQG µSLODXL¶ LV WKH PRVW LPSRUWDQW QXW-producing species in the

Philippines. Canarium luzonicum Miq. most commonly known in the Philippines as

µSLVD¶ DQG µEDVLDG¶ LV LPSRUWDQW QRW DV DQ HGLEOH QXW EXW IRU LWV RLO\ UHVLQ NQRZQ

ORFDOO\DVµVDKLQJ¶ZKLFKLVWDSSHGIURPWKHWUXQN:KHQSURFHVVHGLWLVFDOOHGµEUHD

EODQFD¶ZKLWH Sitch) and is exported as Manila elemi Canarium indicum L. is an

important nut-SURGXFLQJVSHFLHVLQWKH6RORPRQ,VODQGVORFDOO\FDOOHGµQJDOL¶3DSXD

1HZ*XLQHDORFDOO\FDOOHGµJDOLS¶DQG9DQXDWXZKHUHLWLVNQRZQDVµQDQJDL¶ [10].

Canarium album (Lour) Raeusch.NQRZQLQ(QJOLVKDV&KLQHVHROLYHµVDPRFKHHQ¶

LQ7KDLODQGDQGµWUDPWUDQJ¶LQ9LHWQDPLVLPSRUWDQWLQWKHVHFRXQWULHVIRULWVHGLEOH

pulp and kernel [13].

18

Table 1.6 Secondary metabolites of Canarium L.

Compound name Species Chemical

formula

Structure

number

Plant part References

CARBOXYLIC ACIDS Octyl acetate

Canarium schweinfurthii Engl.

C10H20O2

1

resin,oil

[20]

2,5-Dimethoxytoluene Canarium schweinfurthii Engl.

C9H12O2 2 resin,oil [20]

COUMARINS Scoparone

Canarium album (Lour.) Raeusch

C11H10O4

3

fruit

[41]

Scopoletin Canarium album (Lour.) Raeusch

C10H8O4 4 fruit [41]

HETEROCYCLIC COMPOUNDS-FURANS 2-acetylfuran


C6H6O2

5

resin,oil

[20]

n-octanol Canarium schweinfurthii Engl.

C8H18O 6 resin,oil [20]

LIPIDS Hexadecanoic acid


C16H32O2

7

fruit,oil

[42]

9-octadecenoic acid Canarium schweinfurthii Engl.

C18H34O2 8 fruit,oil [42]

6,9-octadecadienoic acid



9,12,15-octadecatrienoic acid



Oleic acid Canarium schweinfurthii Engl.


Linoleic acid Canarium schweinfurthii Engl.


n-decanol Canarium schweinfurthii Engl.

C10H22O 13 resin, oil [20]

n-dodecanol Canarium schweinfurthii Engl.


PHENOLS- FLAVONOIDS Luteolin


C15H10O6

15

fruit

[18]

Luteolin-7-O-ȕ-D-glucoside Canarium album (Lour.) Raeusch

C21H20O11 16 fruit [18]

Quercetin Canarium album C15H10O7 17 fruit [18]

19

Table 1.6 Secondary metabolites of Canarium L. (continuation)


formula

Structure

number


Quercetin-3-O-ȕ-D-glucoside Kaempferol ¶¶-tetrahydroxyflavanone

Canarium album (Lour.) Raeusch Canarium album (Lour.) Raeusch Canarium album (Lour.) Raeusch

C21H19O12 C15H10O6

C15H10O6

18 19 20

fruit fruit fruit

[18] [18] [18]

3,5,7,3'-tetrahydroxy-4'-methoxyflavanonol


C15H10O6 21 fruit [18]

Hyperin/ Quercetin-3-galactoside


C21H20O12 22 dried stem, leaf,fruit

[24] [17]

Kaempferol-3-glucoside Canarium album (Lour.) Raeusch

C21H20O11 23 fruit [17]

Amentoflavone Canarium album (Lour.) Raeusch

C30H18O10 24 fruit [17]

Agathisflavone Canarium manii King. C30H18O10 25 stem,bark [29] PHENOLIC ACIDS Sinapic acid


C11H12O5

26

fruit

[34]

Corilagin Gallic acid

Canarium album (Lour.) Raeusch Canarium album (Lour.) Raeusch

C27H22O18 C7H6O5

27 28

fruit fruit

[41] [17]

Ellagic acid Canarium album (Lour.) Raeusch

C14H6O8 29 dried stem,leaf

[24]

Brevifolin carboxylic acid Canarium album (Lour.) Raeusch

C13H8O8 30 fruit [34]

3-O-galloyl quinic acid butyl ester


C18H24O10 31 fruit [33]

3,4-dihydroxybenzoic acid ethyl ether


C9H10O4 32 fruit [18]

2-hydroxybenzoic acid Canarium album (Lour.) Raeusch


TANNINS Ethyl gallate


C9H10O5

34

fruit

[18] [17]

Methyl gallate Canarium album (Lour.) Raeusch


Elemicin Canarium commune L. C12H16O3 36 fruit

[40]

20



formula

Structure

number


SAPONINS- HYDROXY ACIDS Elemadienonic acid

Canarium boivinii Engl. Canarium schweinfurthii Engl.

C30H46O3

C30H46O3

37 37

resin resin

[44] [32]

TERPENES- CYCLOHEXANE Limonene Furfuryl butanoate

Canarium schweinfurthii Engl. Canarium boivinii Engl. Canarium zeylanicum (Retz.) Blume . Canarium schweinfurthii Engl.

C9H12O3

C9H12O3

38 39

resin,oil resin oleoresin resin, oil

[20] [36] [44] [20]

MONOTERPENES Į-pinene


C10H16

40

resin

[35]

ȕ-Pinene Canarium album (Lour.) Raeusch

C10H16 41 resin [35]

Myrcene Canarium album (Lour.) Raeusch

C10H16 42 resin [35]

Į-Fenchene Canarium album (Lour.) Raeusch

C10H16 43 resin [35]

p-1-Menthene Canarium album (Lour.) Raeusch

C10H18 44 resin [35]

¨-3-Carene Canarium album (Lour.) Raeusch

C10H16 45 resin [35]

Į-Terpinene Canarium album (Lour.) Raeusch

C10H16 46 resin [35]

cis-Sabinene hydrate Canarium album (Lour.) Raeusch

C10H18O 47 resin [35]

Terpinolene Canarium album (Lour.) Raeusch

C10H16 48 resin [35]

Linalool Canarium album (Lour.) Raeusch Canarium schweinfurthii Engl.

C10H18O 49 resin resin,oil

[35] [20]

21



formula

Structure

number


cis-p-Menth-2-en-1-ol


C10H18O

50

resin

[35]

trans-p-Menth-2-en-1-ol Canarium album (Lour.) Raeusch

C10H18O 51 resin [35]

Terpinen-4-ol Canarium album (Lour.) Raeusch

C10H18O 52 resin [35]

Į-Terpineol Canarium album (Lour.) Raeusch Canarium schweinfurthii Engl.

C10H18O 53 resin resin,oil

[35] [20]

cis-Piperitone Canarium album (Lour.) Raeusch

C10H16O2 54 resin [35]

Isobornyl acetate Canarium album (Lour.) Raeusch

C12H20O2 55 resin [35]

1,8-cineole Canarium schweinfurthii Engl.

C10H18O 56 reisn,oil [20]

Citronellyl acetate Canarium schweinfurthii Engl.


Neryl acetate Canarium schweinfurthii Engl.


Decyl Acetate Canarium schweinfurthii Engl.


a-pinene/ Į-pinene

Canarium boivinii Engl. Canarium zeylanicum (Retz.) Blume

C10H16 40 Resin Oleoresin, timber

[36] [44]

Carvone Canarium zeylanicum (Retz.) Blume

C10H14O 60 oleoresin [36]

Į-phellandrene Canarium zeylanicum (Retz.) Blume

C10H16 61 oleoresin, bark,timber

[36]

ȕ-phellandrene Canarium zeylanicum (Retz.) Blume

C10H16 62 oleoresin, bark,timber

[36]

Terpineol Canarium zeylanicum (Retz.) Blume

C10H18O 63 oleoresin, bark

[36]

TRITERPENES Į-amyrin/ a-amyrin/ (urs-12-en-ȕ-ol)

Canarium album (Lour.) Raeusch Canarium boivinii Engl. Canarium zeylanicum (Retz.) Blume

C30H50O

64

dried stem, leaf Resin, oleoresin, bark,timber

[30] [36] [44]

22



formula

Structure

number


ȕ-amyrin(olean-12-en-ȕ-ol)

Canarium album (Lour.) Raeusch Canarium boivinii Engl. Canarium zeylanicum (Retz.) Blume

C30H50O

65

dried stem, leaf Resin oleoresin, bark,timber

[30] [36] [44]

3-epi-Į-amyrin Canarium album (Lour.) Raeusch

C30H50O 66 dried stem, leaf

[30]

3-epi-ȕ-amyrin Canarium album (Lour.) Raeusch

C30H50O 67 dried stem, leaf

[30]

Į-Amyrenone (urs-12-en-3-one)

Canarium zeylanicum (Retz.) Blume


ȕ-Amyrenone (olean-12-en-3-one)



Taraxerol Canarium zeylanicum (Retz.) Blume

C30H50O 70 bark [36]

urs-12-ene-Įȕ-diol Canarium album (Lour.) Raeusch

C30H50O2 71 dried stem,leaf

[30]

olean-12-ene-ĮȕGLRO Canarium album (Lour.) Raeusch

C30H50O2 72 dried stem, leaf

[30]

TETRATERPENES CAROTENOIDS All-trans-lutein

Canarium odontophyllum Miq.

C40H56O2

73

peel,pulp, seed

[26]

9-cis-lutein Canarium odontophyllum Miq.

C40H56O2 74 peel,pulp, seed

[26]

13-cis-lutein Canarium odontophyllum Miq.

C40H56O2 75 peel,pulp, seed

[26]

15-cis-ȕ-carotene Canarium odontophyllum Miq.

C40H56 76 peel,pulp, seed

[26]

9-cis-ȕ-carotene Canarium odontophyllum Miq.


[26]

All-trans-ȕ-carotene Canarium odontophyllum Miq.


[26]

SESQUITERPENES Į-Cubenene


C15H24

79

resin

[35]

Į-Copaene Canarium album (Lour.) Raeusch

C15H24 80 resin [35]

23



formula

Structure

number


ȕ-Cubebene


C15H24

81

resin

[35]

Canaric acid Canarium muelleri F.M. Bailey Canarium zeylanicum (Retz.) Blume

C30H48O2 82 oleoresin [36] [37]

(E)-ȕ-Caryophyllene Canarium album (Lour.) Raeusch

C15H24 83 resin [35]

Į-Humelene Canarium album (Lour.) Raeusch

C15H24 84 resin [35]

Germacrene D Canarium album (Lour.) Raeusch

C15H24 85 resin [35]

Spathulenol Canarium album (Lour.) Raeusch Canarium schweinfurthii Engl.

C15H24O 86 Resin resin,oil

[20] [35]

Viridiflorol Canarium schweinfurthii Engl.

C15H26O 87 resin, oil [20]

Caryophyllene epoxide Canarium album (Lour.) Raeusch

C15H24O 88 resin [35]

(E)- Nerolidol Canarium schweinfurthii Engl.


Elemene Canarium zeylanicum (Retz.) Blume

C15H24 90 oleoresin, bark

[36]

Elemol Canarium zeylanicum (Retz.) Blume

C15H26O 91 oleoresin, bark

[36]

Brevifolin Canarium album (Lour.) Raeusch

C10H12O4 92 dried stem, leaf

[24]

į-Elemene Canarium album (Lour.) Raeusch

C15H24 93 resin [35]

STEROLS Sitosterol


C29H50O

94

oleoresin, bark,timber

[36]

ȕ-sitosterol Canarium benglense Roxb.

C29H50O 95 bark [45]

1HRLOH[RQROȕ-Hydroxyurs-12-en-11-one)


C30H48O2 96 oleoresin, bark

[36]

24

1.

2.

3.

4.

5.

6.

7.

8.

9.

Figure 1.1: Chemical structures of secondary metabolites from Canarium L.

25

10.

11.

12.

13.

14.

15.

16.


26

17.

18.

19.

20.

21.


27

22.

23.

24.


28

25.

26.

27.

28.


29

29.

30.

31.

32.

33.


30

34.

35.

36.

37.

38.


31

39.

40.

41.

42.

43.

44.

45.


32

46.

47.

48.

49.

50.

51.


33

52.

53.

54.

55.

56.

57.

58.


34

59.

60.

61.

62.

63.

64.


35

65.

66.

67.

68.


36

69.

70.

71.

72.


37

73.

74.

75.

76.

77.


38

78.

79.

80.

81.

82.


39

83.

84.

85.

86.

87.


40

88.

89.

90.

91.

92.

93.


41

94.

95.

96.


42

In Melanesia, marketing operations for Canarium indicum L. and Canarium harveyi

Seem. range from private and community based production, processing, and

marketing of kernel oil for cosmetic and medicinal use, to government-backed

nationwide purchasing of kernels for sale as confections and oil. Canarium

solomonense B.L. Burtt in the Solomons islands are used as general-purpose timber

[46]. Because of the potential of Canarium L. as a high-value export crop for nuts (for

confections) and/or oil extraction, some research is being done on its taxonomy,

production and marketing. Additional research needs include selection, evaluation,

and improvement of promising varieties for timber production, investigation of

cultural aspects, phenological studies, and vegetative propagation [47].

1.7 Selection of plant material

The selection of plant material for the screening of biological activity can be based on

a random selection or based on ethnopharmacology, where existing knowledge of the

particular healing properties have been handed down from generation to generation

especially amongst traditional healers. An additional mechanism for the identification

of the plants for the study of its chemical constituents is based on chemotaxonomy.

Chemotaxonomy is a science focusing on the correlation between related plant

species and the occurrence of similar secondary metabolites.

43

1.7.1. The selection of Canarium patentinervium Miq.

Despite reasonable traditional use of Canarium L. species, as well as extreme

botanical diversity, the Canarium species in the Malaysian region have neither been

the subject of pharmacological nor extensive phytochemical studies. Claims of the

traditional usage to heal wounds [12] therefore requires validation and accurate

documentation. For this purpose, this study was initiated as a basis for scientific

verification regarding the traditional use of Canarium patentinervium Miq. . The

leaves and barks of Canarium patentinervium Miq. were collected from one

individual tree in April 2010 from Bukit Putih, Selangor, Malaysia (3°5'24 "N

101°46'0"E). The plant was identified by Mr. Kamaruddin (Forest Research Institute

of Malaysia). A herbarium sample (PID 251210-12) has been deposited in the Forest

Research Institute of Malaysia.

1.7.2 The selection of bioassays performed

The origin and design of a screening process incorporates knowledge attained in

ethnomedicine, traditional uses of the plant species, phytochemical evaluation and

correlation to specific biological targets as well as the use of natural product libraries

and general or targeted literature review. Stable standardized crude extracts are

prepared and assayed for the claimed activities for which the particular plant species

is traditionally used. The present study is focused on antioxidant, anti-inflammatory,

anti-acetylcholinesterase, antimicrobial, antiparasitic and anticancer activities.

44

1.8 Aims of study

This study is undertaken to extract and to isolate and purify active constituents from

plants by phytochemical techniques and to characterize, elucidate and identify the

structure of the pure compound and to assay isolated plant active constituents for

antibacterial, antiparasitic, antioxidant, anti-inflammatory, anti-acetylcholinesterase

and anticancer activities (Figure 1.2).

1.9 Objectives of the study

i. Extraction of leaves and barks of plant with solvents of different polarity to

obtain crude extracts.

ii. Investigation of the secondary metabolites present in the crude extracts of

leaves and barks by qualitative phytochemical analysis.

iii. Investigation of the antioxidant activity of the crude extracts of both leaves

and barks using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging

assay, ferric reducing ability of plasma (FRAP) assay, ¶-Azino-bis (3-ethyl-

benzthiazoline-6-sulfonic acid) (ABTS) assay, ȕ-carotene bleaching assay and

superoxide dismutase assay (SOD).

iv. Investigation of the anti-inflammatory potential of the crude extracts against 5-

lipoxygenase (5-LOX) and cycloxygenase (COX) systems in vitro.

v. Investigation of the anti-acetylcholinesterase (AChE) potential of the crude

extracts in vitro.

45

vi. Investigation of the in vitro antibacterial activity of the crude extracts of leaves

and barks using the disc diffusion assay and determination of minimum

inhibitory concentration, minimum bactericidal concentration and death kill

rate against four ATCC bacterial strains namely Gram-positive bacteria

Staphylococcus aureus, Bacillus cereus, methicillin-resistant Staphylococcus

aureus and Gram-negative Pseudomonas aeruginosa and nine clinical strain

bacteria.

vii. Investigation of the in vitro antifungal activity of the crude extracts of leaves

and barks using the disc diffusion assay against three clinical strain fungi

namely, Candida parapsilosis, Candida glabrata and Candida albicans.

viii. Investigation of the anticancer activity of the crude extracts of the leaves and

barks against human derived cell lines [HCT 116,colon cancer cell line and

MCF-7 (ER+), MDA 468 (ER-) breast carcinoma] using MTT assay.

ix. Investigation of the in vitro anti-parasitic activity of the crude extracts of

leaves and barks against Leishmania donovani promastigotes, Entamoeba

histolytica and Giardia intestinalis.

x. Isolation and identification of the chemical compounds responsible for the

antibacterial and antioxidant activity in the most active extract through

bioassay-guided fractionation using liquid-liquid extraction or solvent

partioning, column chromatography, thin layer chromatography (TLC) and

nuclear magnetic resonance (NMR).

xi. Bioactivity screening of all isolated compounds using the same bioassay as

mentioned in iii-viii.

46

Leaves and barks of Canarium patentinervium

Miq.

Succesive maceration extraction with solvent hexane, chloroform and

ethanol

Hexane extract

Chloroform extract

Ethanol extract

Bioassay ( in vitro)

Most active extract

Preliminary fractionation with solvent partioning and

column chromatography

Bioassay ( in vitro)

Active fraction(s)

Different chromatographic

techniques for isolation and purification of

compound(s) guided by in vitro bioassay

Active compound (s)

Structure determination by NMR

Bioassay of pure compound (s) using different concentrations

to determine potency

Figure 1.2: A diagrammatic summary of steps in the study of Canarium patentinervium Miq. evaluating the phytochemistry and pharmacological activities

47

CHAPTER 2

PLANT COLLECTION AND EXTRACTION

2.1 Brief introduction to Canarium patentinervium Miq.

Canarium patentinervium Miq. is fairly widespread species which occurs in lowland

primary and secondary mixed dipterocarp forest and kerangas forest. It is recorded as

a common understorey tree,W¶VDVPDOOWRPHGLXP-sized tree reaching a height of 50-

60 feet. The bark is grey brown that is smooth to cracking with many small lenticels

and often inconspicuous hoop marks. The inner bark is reddish brown and loosely

fibrous with clear resinous exudate. The sapwood is whitish with strong resinous

smell from slash. Stipules are caduceus, inserted at the junction of twigs and rachis.

Leaves are about 6-15 inches long and thickened at both ends, blade ovate to oblong

lanceolate. Apex is gradually to rather shortly and bluntly acuminate. Fruits are

ellipsoid to obovoid, rounded with a stone triangular in cross section. Its¶ strongly

glaucous and strongly aromatic (Figure 2.1). Tree flowers mainly from November to

May and fruits from September to April [14].

2.2 Plant collection and extraction

The leaves and barks of Canarium patentinervium Miq. were collected from one

individual tree in April 2010 from Bukit Putih, Selangor, Malaysia (3°5'24 "N

101°46'0"E). The plant was identified by Mr. Kamaruddin (Forest Research Institute

48

Figure 2.1: Fresh and dried herbarium sample of Canarium patentinervium Miq.

49

of Malaysia). A herbarium sample (PID 251210-12) has been deposited in the Forest

Research Institute of Malaysia. The leaves and barks were air dried at ambient

temperature (25 oC) in the laboratory for about 15 days. Thereafter the leaves were

separated from the barks and any fruit present were removed and stored separately.

The plant material was crushed to a fine powder using an industrial grinder.

Extraction was performed with maceration that involves leaving the pulverized plant

to soak in a suitable solvent in a closed container at room temperature [48]. Dried and

grinded sample of leaves (2.8 kg) and barks (1.7 kg) were soaked in hexane with the

ratio of 1:3 parts of sample to solvent for 2 hr in a 60 oC water bath, then filtered and

concentrated with a rotary evaporator (Buchi, R-200 Switzerland). This was repeated

3 times. Thereafter the leaves and barks were left to air dry completely for 3 days

before repeating the whole process with chloroform and then ethanol respectively.

The yield for the hexane, chloroform and ethanol extract of leaves were 1.25 %, 1.11

% and 6.45 % respectively. The yield for the hexane, chloroform and ethanol extract

of barks were 1.04 %, 0.40 % and 2.61 %. Crude extracts were kept at -20 oC until

further use.

50

CHAPTER 3

PHYTOCHEMICAL METHODS

3.1 Introduction

Phytochemical screening is basically done to detect the types of secondary

metabolites that may be present in the plant extract. A positive reaction should not be

taken as proof of presence of a certain type of secondary metabolite because other

compound types may give false-positive reactions. Despite this caveat, these detection

methods are often effective for generating hyphotheses about what types of secondary

PHWDEROLWHV PD\ EH SUHVHQW LQ D PL[WXUH RI ³XQNQRZQV´ DQG RI PRQLWRULQJ WKH

presence of compounds of interest [48]. Qualitative phytochemical analysis of the

crude extract was determined as outlined below.

3.2 Phytochemical assay protocol

Qualitative phytochemical analysis of the crude extract was determined as outlined

below [48][49].

i. Alkaloids test

200 mg of the extract was dissolved in 10 ml of methanol and heated on a

boiling water bath with 2M HCl (5 ml). After cooling, the mixture was

filtered and the filtrate was divided into two equal portions. One portion

was treated with a few drops of Mayer's reagent and the other with equal

51

amounts of Wagner's reagent. The samples were then observed for the

presence of turbidity or precipitation. A (+) score was recorded if the

reagent produced only a slight opaqueness; a (++) score was recorded if a

definite turbidity, but no flocculation was observed and a (+++) score was

recorded if a definite heavy precipitate or flocculation was produced.

0D\HU¶VUHDJHQWFRQVLVWVRIVROXWLRQVGHVFULEHGKHUHDVVROXWLRQ,DQGVROXWLRQ,,

Solution I: 1.36 g HgCl2 was dissolved in 60 ml water. Solution II: 5 g KI was

dissolved in 10 ml water. Both solutions are combined and diluted with water to 100

ml. If alkaloids are present a white to yellowish precipitate will appear. Wagner

reagent consists of 1.27 g I2 (sublimed) and 2 g KI was dissolved in 20 ml water and

water is made up to 100 ml. A brown precipitate indicates the presence of alkaloids.

ii. Flavonoids test

40 mg plant material was dissolved in 2 ml ethanol and filtered. The

filtrate was treated with a few drops of concentrated HCl and magnesium

turnings (0.5 g). The presence of flavonoids was indicative if pink or

magenta-red color developed within 3 min.

iii. Saponins test

About 2.5 g of the plant material was extracted with boiling water. After

cooling, the extract was shaken vigorously to froth and was then allowed

to stand for 15-20 min and classified for saponin content as follows: (no

froth = negative; froth less than 1 cm = weakly positive; froth 1.2 cm high

= positive; and froth greater than 2 cm high = strongly positive).

52

iv. Tannins test

About 10 mg of extract was dissolved in 6 ml of hot distilled water and

filtered. The solution is divided in three test tubes. To the first 0.9 %

sodium chloride solution was added, to the second 0.9 % sodium chloride

and 1 % gelatine solution was added and to the third ferric chloride (FeCl3)

was added. Formation of a precipitate in the second treatment suggests the

presence of tannins, and a positive response after addition of FeCl3 to the

third portion which will result in a characteristic blue, blue-black, green or

blue-green color supports this inference.

v. Sterols (Salkowski reaction) test.

40mg of extract was dissolved in 2 ml of chloroform and filtered. The

filtrate was then added to 1 ml of concentrated H2SO4. The presence of

sterols was indicated by the 2 phase formation with a red color in the

chloroform phase.

3.3 Results

Phytochemical analysis results exhibits the presence of flavonoid, tannin and sterols

in the respective leaves and barks extracts as shown in Table 3.1. Presence of

flavonoid and tannin was shown in the leaf and bark extract of ethanol (Figure 3.1 and

3.2). Steroids were present in all extracts except leaf extract of hexane and chloroform

(Figure 3.3 and Figure 3.4).

53

3.4 Discussion

Phytochemical analysis of Canarium patentinervium Miq.(Table 3.1) revealed

presence of tannins and flavonoids in the ethanol extract of leaves and barks.

Triterpenoids can be divided into at least four groups of compounds namely true

triterpenes, steroids, saponins and cardiac glycosides [50]. Steroids were detected in

the ethanol extract of leaves and all tested bark extracts.

3.5 Conclusion

Results from our phytochemical analysis revealed that the ethanol extract of leaves

and barks of Canarium patentinervium Miq. accumulate substantial amounts of

flavonoids and tannins which could be possibly result in antioxidant or antibacterial

activity [51].

54

Table 3.1: Preliminary phytochemical analysis of the crude extract of Canarium patentinervium Miq

Sample Alkaloids Flavonoids Saponins Tannins Steroids

LH - - - - -

LC - - - - -

LE - +++ - +++ +++

BH - - - - +++

BC - - - - ++

BE - +++ - +++ +++

LH-leaf hexane extract, LC- leaf chloroform extract, LE-leaf ethanol extract, BH- bark hexane extract, BC- bark chloroform extract, BE- bark ethanol extract -:negative, +:trace, ++:positive, +++: strongly positive

55

Figure 3.1: Positive flavonoid result for leaf ethanol extract (left) against blank and and bark ethanol extract (right) against blank.

56

Figure 3.2: Positive tannin result for OHDIHWKDQROH[WUDFWPDUNHGDVWHVWWXEHµ56LE¶OHIW DQGEDUNHWKDQROH[WUDFWPDUNHGDVWHVWWXEHµ6¶ULJKW against

blank (-), positive standard tannic acid(+) and ferric chloride .

57

Figure 3.3: Positive sterol test for leaf ethanol extract (left) and bark ethanol extract (right) against blank.

58

Figure 3.4: Positive sterol test for bark hexane extract (left) and bark chloroforml extract (right) against blank.

59

CHAPTER 4

THE ANTIOXIDANT ACTIVITY

4.1 Introduction

4.1.1 Free radicals and their mechanism of action

Oxygen is important for life processes to occur, however, an excess of oxygen could

result in oxidative damage, which may even lead to death. The damage is not due to

the presence of oxygen, but rather due to its role in the reduction of certain products

to toxic free radicals as natural by products of cell metabolism [52]. These free

UDGLFDOVDUHSURGXFHGZLWKLQ OLYLQJFHOOVDQGDUHSDUWRI WKHFHOO¶V normal metabolic

processes, including detoxification processes and immune system defences. Free

radicals may either be oxygen derived (ROS, reactive oxygen species) or nitrogen

derived (RNS, reactive nitrogen species) [53]. The oxygen derived molecules are O2-

(superoxide), HO (hydroxyl), HO2 (hydroperoxyl), ROO (peroxyl), RO (alkoxyl) as

free radical and H2O2 oxygen as non-radical. Nitrogen derived oxidant species are

mainly NO (nitric oxide), ONOO (peroxy nitrate), NO2 (nitrogen dioxide) and N2O3

(dinitrogen trioxide) [54].

In normal cell, there are appropriate oxidant and antioxidant balance. However this

balance can be shifted, when the production species is increased or when levels of

antioxidants are diminished. This stage is called oxidative stress [53]. Oxidative stress

results in the damage of biopolymers including nucleic acids, proteins,

polyunsaturated fatty acids and carbohydrates. Oxidative stress causes serious cell

60

GDPDJHOHDGLQJWRDYDULHW\RIKXPDQGLVHDVHVOLNH$O]KHLPHU¶VGLVHDVH3DUNLQVRQV¶V

disease, atherosclerosis, cancer, liver disease, inflammation, diabetes, AIDS, arthritis,

immunological incompetence, neurodegenerative disorders and etc [55][56].

In aerobic organisms, the defence system against these free radicals is provided by

free radical scavengers which act as anti-oxidants. Antioxidants are defined as a

molecule capable of slowing or preventing the oxidation of other molecules, whereas

DELRORJLFDODQWLR[LGDQWKDVEHHQGHILQHGDV³DQ\VXEVWDQFHWKDWZKHQSUHVHQWDWORZ

concentrations compared to those of an oxidizable substrate, significantly delays or

SUHYHQWVR[LGDWLRQRIWKDWVXEVWUDWH´[56] . There are at least four general sources of

antioxidants: (1) enzymes, for example super oxide dismutase, gluthathione

peroxidase and catalse; (2) large molecules (albumin, ceruloplasmin, ferritin, other

proteins); (3) small molecules [ascorbic acid, glutathione, uric acid, tocopherol,

carotenoids, (poly)phenols]; and (4) some hormones (estrogen, angiotensin,

melatonin, etc) [54][56].

Mechanisms of antioxidant action include serving as (1) physical barriers to prevent

ROS generation or ROS access to important biological sites, eg UV filters, cell

PHPEUDQHV FKHPLFDO WUDSVVLQNV WKDW ³DEVRUE´ HQHUJ\ DQG HOHFWURQV TXHQFKLQJ

ROS, eg carotenoids and anthocyanidins; (3) catalytic systems that neutralize or divert

ROS, eg SOD (superoxide dismutase), catalse and gluthathione peroxidase; (4)

binding/inactivation of metal ions to prevent generation of ROS, eg ferritin,

ceruloplasmin, catechins; and (5) chain breaking antioxidants which scavenge and

destroy ROS, eg ascorbic acid (vitamin C), tocopherols (vitamin E), uric acid,

gluthathione, flavonoids [57].

61

4.1.2 Natural antioxidants

The term, antioxidant is used to describe a component that can function to decrease

tissue damage by reactive oxygen. Defence provided for by the anti-oxidant systems

is crucial to survival and can operate at different levels within the cells through the

prevention of radical formation, intercepting formed radicals, repairing oxidative

damage, increasing the elimination of damaged molecules, and recognition of

excessively damaged molecules, which are not being repaired but rather eliminated to

prevent mutations from occurring during replication [58]. Non-enzymatic anti-

oxidants are classified as being either water-soluble or lipid-soluble, depending on

whether they act primarily in the aqueous phase or in the lipophilic region of the cell

membranes [51].

The hydrophilic anti-oxidants include vitamin C (ascorbic acid) and certain

polyphenol flavonoid groups, while the lipophilic anti-oxidants include ubiquinols,

retinoids, carotenoids, apocynin, procyanidins, certain polyphenol flavonoid groups

and tocopherols [59]. Other non-enzymatic anti-oxidants include antioxidant enzyme

cofactors, oxidative enzyme inhibitors and transition metal chelators such as ethylene

diamine tetra-acetic acid (EDTA) [58].

62

4.1.3 Therapeutic potential of phenolic substances

The establishment of an inverse correlation between the intake of fruits and

vegetables and the occurrence of diseases such as inflammation, age-related disorders,

cancer and cardiovascular disease is derived from clinical trials and epidemiological

studies [58][59]. Phenolic substances, which are known to possess high antioxidative

activity, are actually common phytochemicals in fruits and leafy vegetables. Plants

containing phenolic compounds have been reported to possess strong antioxidant

properties. Most of these phenolics are classified into two principal groups of phenol;

carboxylic acids and flavonoids, the latter being the most significant [58]. Phenolic

compounds are found abundantly in all parts of the plant, such as wood, bark, stems,

leaves, fruit, root, flowers, pollen and seeds. Antioxidative activity of phenolic

compounds is based on their ability to donate hydrogen atoms to free radicals. Many

phenolic compounds, particularly flavonoids, exhibit a wide range of biological

effects, including antioxidant activity, antibacterial, antiviral, anti-inflammatory,

antiallergic, anticancer, anti-thrombotic, vasodilatory actions and the ability to lower

the risk of coronary heart diseases [58].

Knowledge of the potential antioxidant compounds present within a plant species

does not necessarily indicate its antioxidant capacity, as the total anti-oxidant effect

may be greater than the individual antioxidant activity of one compound, due to

synergism between different anti-oxidant compounds. Methods to measure the anti-

oxidant activity in plant material generally involve both the generation of radicals

(and their related compounds), and the addition of anti-oxidants, the latter resulting in

the reduction of the radical and its consequent disappearance [60][61]. The role of

63

free radicals and active oxygen is becoming increasingly recognized in the

pathogenesis of the many human diseases, including cancer, neurodegenerative

diseases, ageing and atherosclerosis [62].

Free radicals can also cause lipid peroxidation in foods that leads to their

deterioration. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and

butylated hydroxytoluene (BHT), have restricted use in food as they are suspected of

carcinogenicity and of hormonal perturbation [63][64]. In search for sources of novel

anti-oxidants with low toxicity, medicinal plants have over the past few years been

studied extensively for their radical scavenging activity [65]. As plants produce a

large number of anti-oxidants to control the oxidative stress caused by sunbeams and

oxygen, it is clear that plants may represent a source of new compounds with

antioxidant activity [66].

4.1.4 Antioxidant potential of Canarium L.

Antioxidant activities were reported in Canarium album (Lour.) Raeusch, Canarium

odontophyllum Miq. and Canarium schweinfurthii Engl. A tonic soup made of

Canarium album (Lour.) Raeusch used mainly in China displayed significant

antioxidant activity by 1,1,-diphenyl-2-2-picrylhydrazyl (DPPH) and ferric reducing

antioxidant power (FRAP) assay [22]. Tannins extracted from the leaves, twigs and

stem barks of Canarium album (Lour.) Raeusch showed potent antioxidant activity in

the DPPH radical scavenging activity with IC50 values of 56.86 µg/ml, 62.31 µg/ml

and 54.80 µg/ml respectively and ferric reducing power of 4.28, 3.74 and 4.49 mmol

AAE/g equivalent of dried tannin [67]. Pure compounds from this species includes

64

brevifolin, hyperin and ellagic acid which showed free radical scavenging activity in

DPPH assay [24]. The essential oil of Canarium schweinfurthii Engl. was tested for

WKH DQWLR[LGDQW DFWLYLW\ ZLWK WKH '33+ DVVD\ DQG E\ ȕ-carotene bleaching test. It

exhibited highest antioxidant activity at 150 µg/ml activity in both assay [25]. The

ethyl acetate fraction of peel of Canarium odontophyllum Miq. exhibited 95±1.00%

scavenging activity at the concentration of 40 µg/ml [26][68].


4.2.1 Antioxidant determination assays

Major antioxidant capacity methods have been generally divided into two categories:

(1) hydrogen atom transfer (HAT) reaction and (2) single electron transfer (SET)

reaction-based method. SET and HAT almost always occur together with the balance

determined by antioxidant structure and pH. Bond dissociation energy (BDE) and

ionization potential (IP) are two major factors that determine the mechanism and the

efficacy of the antioxidants. HAT-based methods measure the classical ability of an

antioxidant to quench free radicals by hydrogen donation. Relative reactivity in HAT

methods are determined by the BDE of the H-donating group in the potential

antioxidant and IP. Most HAT-based methods monitor competitive reaction kinetics

and are rapid.

65

;$+ ;+$

SET-based methods detect the ability of a potential antioxidant to transfer one

electron to reduce any compound, including metals, carbonyls and radicals.

;$+ X- $+

Relative reactivity is based on deprotonation and IP of the reactive functional group

so SET is pH dependent. In general, IP decrease with increasing pH, reflecting

increased electron-donating capacity with deprotonation. At acidic conditions, the

reducing capacity may be restrained due to protonation on antioxidant compounds,

whereas in basic conditions, proton dissociation of phenolic compounds would

increase samples reducing capacity. SET methods can be relatively slow and involve

two components, antioxidant and oxidant (probe).

Probe (oxidant) + e (from antioxidant) reduced probe + oxidised antioxidant

Huang et al. [69] broadly classified antioxidant capacity assays based on the reaction

mechanisms as follows : SET [Folin-Ciocalteu (Folin-C), 1,1-diphenyl-2-

picrylhydrazine (DPPH) radical scavenging, trolox equivalent antioxidant capacity

(TEAC), ferric reducing ability of plasma (FRAP)]; and HAT [oxygen radical

absorbance capacity (ORAC), ȕ-carotene/linoleic acid model system and inhibition of

phospholipid peroxidation]. Prior et al. [70] classified DPPH, TEAC and Folin-C

antioxidant assays into a category that utilises both SET and HAT reaction

mechanisms, though predominantly based on electron transfer [70][53].

66

4.2.2 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay


The antioxidant activity of each of the plant extracts was determined using the

colorimetric DPPH assay, as described by Juan Badaturuge [71], was employed to

determine the radical scavenging activity of the plant extracts. In DPPH radical-

scavenging assay, antioxidants react with DPPH, and convert it to yellow coloured

ĮĮ-diphenyl-ȕ-picryl hydrazine. The degree of discolouration indicates the radical-

scavenging potential of the antioxidant activities [72] (Figure 4.1). The hydrogen

donating capacity of test samples is quantified in terms of their ability to scavenge the

relatively stable, organic free radical DPPH and by consequent reduction thereof. The

absorption of the deep violet DPPH solution is measured at 550 nm, after which

absorption decreases due to decolourisation to a yellow-white colour, in the event of

reduction [73]. This decrease in absorption is stoichiometric according to the degree

of reduction. The remaining DPPH is measured at a time interval of 30 min after the

addition of the DPPH, which corresponds inversely to the radical scavenging activity

of the sample extract or anti-oxidant.

67

FIOH ± flavonoid compound, FIO. ± flavonoid having donated a hydrogen

Figure 4.1: Diagrammatic representation of chemical reaction of the reaction of DPPH in the presence of an electron donating antioxidant [74].

DPPH relatively stable

organic radical ± deep violet colour

DPPH reduced in presence of anti-

oxidant ± yellow-white colour

68


Aliquots of plant extract dissolved in dimethyl sulfoxide (DMSO, R&M) were plated

out in triplicate in a 96-well microtiter plate at different concentrations, prepared as

serial dilutions ranging from 10 µg/ml to 0.3125 µg/ml. The 0.1 mM DPPH solution

(AldrichR) was added to alternating columns of the test samples and methanol for

control of test samples, in the remaining columns. The plate was shaken for 2 min and

incubated for 30 min in the dark. The percentage decolourisation was obtained

spectrophotometrically at 550 nm using the Thermo Scientific Varioskan Flash

microtiter plate reader, linked to a computer equipped with (SkanIt Software 2.4.3).

Percentage decolourisation was plotted against the concentration of the sample and

the EC50 values were determined using Prism 5.00 software. Vitamin C (l-ascorbic

acid), quercetin and trolox was used as positive control. At least three independent

tests were performed for each sample. The DPPH absorbance decreases with an

increase in DPPH radical scavenging activity. This activity is given as percent DPPH

radical scavenging, which is calculated with the equation:

DPPH radical scavenging activity (%) = [(Abscontrol - Abssample)]/ (Abscontrol)] x

100

Abscontrol is the absorbance of DPPH radical + methanol;

Abssample is the absorbance of DPPH radical + sample extract /standard.

69

4.2.3 ¶-Azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) assay



colorimetric ABTS assay, as described by Miller et al. [75], Rice-Evans [76] and

Roberta et al. [77] was employed to determine the radical scavenging activity of the

plant extracts. The ABTS anti-oxidant assay, also known as the Trolox equivalent

anti-oxidant capacity (TEAC) assay, assesses the total radical scavenging capacity of

the plant extracts. This is determined through the ability of these extracts to scavenge

the long-lived specific ABTS radical cation chromophore in relation to that of Trolox,

the water-soluble analogue of vitamin E [78]. The generation of the ABTS+

blue/green chromophore occurs through the oxidation of ABTS diammonium salt in

the presence of potassium persulfate (Figure 3.4), with the absorption maxima

occurring at wavelengths 645 nm, 734 nm and 815 nm. Anti-oxidants will reduce the

pre-formed radical cation to ABTS, and in so doing bring about the decolourisation of

ABTS+ to a colourless product [76]. The extent of this decolourisation is a measure of

the ABTS+ radical cation that has been scavenged, after a fixed time period, and is

relative to the Trolox standard.

70

Figure 4.2: Diagrammatic representation of the formation of the ABTS radical after the addition of potassium persulphate.

ABTS radical

relatively stable

organic radical- deep

turquoise green

ABTS- transparent

71



out in triplicate in a 96-well microtiter plate at different concentrations. At least three

independent tests were performed for each sample. Trolox (6-hydroxy-2,5,7,8-

tetramethylchromon-2-carboxylic acid, Sigma-Aldrich) Vitamin C (l-ascorbic acid)

and quercetin was used as positive control and prepared in ethanol and serial

dilutions of this positive control were also prepared. Ethanol was used as the negative

control. The stock solution included 7 mM ABTS solution and 2.4 mM potassium

persulfate solution. The working solution was then prepared by mixing the 2 stock

solutions in equal quantities. This solution was then stored in the dark for 12 - 16

hours in order to stabilise it before use. It remains stable for 2 - 3 days in the dark.

The concentrated ABTS+ solution was diluted with cold ethanol shortly before

conducting the assay, to a final absorbance of 0.70 ± 0.01 at 734 nm at 37 °C, in a 3

cm cuvette. The total scavenging capacity of the extracts was quantified through the

addition of 100 ȝO$%76to 100 ȝORISODQWH[WUDFW7KHVROutions were heated to 37

°C for 7 min, after which the absorbance was read at 734 nm on a spectrophotometer

(Thermo Scientific Varioskan Flash microtiter plate reader, linked to a computer

equipped with SkanIt Software 2.4.3). All experiments were done in triplicate. The

percentage decolourisation was calculated using equation below and the extent of

inhibition of the absorbance of the ABTS+ was plotted as a function of the

concentration.

72

This activity is given as percent ABTS radical scavenging, which is calculated with

the equation:

ABTS radical scavenging capacity (%) = [(Abscontrol - Abssample)]/ (Abscontrol)] x

100

Abscontrol is the absorbance of ABTS radical +ethanol;

Abssample is the absorbance of ABTS radical + sample extract /standard.

4.2.4. Ferric reducing ability of plasma (FRAP) assay

.



colorimetric FRAP assay, as described by Benzie & Strain [79] with slight

modifications. The mechanism associated with this method is shown in Figure 4.3.

This method was first developed to quantitate ascorbic acid in serum or plasma. When

a Fe3+-TPTZ complex is reduced to the Fe2+ form by an antioxidant under acidic

conditions, an intense blue color with maximum absorption develops at 593nm.

Therefore, the antioxidant effect (reducing ability) can be evaluated by monitoring the

formation of a Fe2+-TPTZ complex with a spectrophotometer. The FRAP assay gives

fast and reproducible results.

73

Figure 4.3: Formation of (Fe2+-TPTZ) complex from (Fe3+-TPTZ) complex by antioxidant.

74



out in triplicate in a 96-well microtiter plate at different concentrations. Vitamin C (l-

ascorbic acid), quercetin and trolox were used as positive control. At least three

independent tests were performed for each sample. The working FRAP reagent was

prepared just before assay by mixing 300 mM of acetate buffer (pH 3.6), 10 mM of

2,4,6-tripyridyl-s-triazine (TPTZ) and 20 mM of FeCl3.6H2O in ratio of 10:1:1.

Briefly 180 µl of the FRAP reagent was mixed with 20 µl of the test sample so that

the final dilution of the test sample in the reaction mixture was 1/10. After 30

minutes, the absorbance of the coloured product (ferrous triphridyltriazine complex)

was recorded using a spectrophotometer (Thermo Scientific Varioskan Flash

microtiter plate reader, linked to a computer equipped with SkanIt Software 2.4.3). Fe

(II) concentrations in the range of 1 µM-100 µM (FeSO4.7H2O) was used as standard

for calibration curve and equation of linearity is determined (y=ax+b). From the

linearity equation, concentration of sample that produced same absorbance as 1mM of

Fe (II) was determined (y of sample filled in equation to obtain x). The antioxidant

activity was calculated as Ferrous Equivalents, the concentration of samples which

produced an absorbance value equal to that of 1 mM FeSO4.

75

4.2.5 ȕ-carotene bleaching assay


7KHȕ-carotene bleaching assay was conducted according to the method described by

Habtemariam et al. [80] with some modifications. ,W KDV ORQJ EHHQ NQRZQ WKDW ȕ-

FDURWHQHUHDFWVZLWKWKHSHUR[\UDGLFDOWRSURGXFHWKDWȕ-carotene epoxides. Therefore

that ȕ-carotene has received attention as a radical scavenger or antioxidant. Later an

DQWLR[LGDQW DVVD\ XVLQJ WKDW ȕ-carotene combined with lipids such as linolenic acid

was established [80]. As shown in Figure 4.4, lipids such as linolenic acid forms as

SHUR[\UDGLFDO/22LQSUHVence of the ROS and O2. This peroxy radical reacts with

ȕ-carotene to form a stable ȕ-FDURWHQHUDGLFDOVXEVHTXHQWO\WKHDPRXQWRIȕ-carotene

reduces in the testing solution. If an antioxidant is present in the testing solution, it

reacts competitively with the peroxy radical. Therefore, antioxidant effects are easily

monitored by the bleaching of the colour of a test solution with a spectrophotometer at

470 QPZKLFKLVWKHW\SLFDODEVRUEDQFHE\ȕ-carotene [56].

ȕ-carotene /22ȕ-carotene adduct

/22

Antioxidant /22ȕ-carotene adduct

Figure 4.4: )RUPDWLRQRIDGGXFWVIURPE\ȕ-carotene and antioxidant with lipid

peroxide radical.

76



out in triplicate in a 96-well microtiter plate at different concentrations. Vitamin C (l-

ascorbic acid), quercetin and trolox were used as positive control. %ULHIO\PORIDȕ-

carotene solution in chloroform (2 mg in 10 ml) was pipetted into a round bottom

flask containing 40 µl of linoleic acid and 500 µl of Tween 20. After the removal of

chloroform using a rotary vacuum evaporator at 45 oC, 100 ml of deionised water

were added with vigorous agitation. 180 µl of the emulsion was added to 20 µl of test

samples at varying concentrations in 96-well microtitre plate. The absorbance was

measured at 470 nm immediately against a blank consisting of the emulsion without

ȕ-carotene and after 3 hr of incubation at 50 oC using a spectrophotometer (Thermo

Scientific Varioskan Flash microtiter plate reader, linked to a computer equipped with

SkanIt Software 2.4.3). All determinations were carried out in triplicates.

The antioxidant activity of test agents was evaluated in terms of bleaching RI ȕ-

carotene using the following formula:

Antioxidant activity AA (%) = [1- (A0-At)/(Aoƍ-Atƍ@[

A0 and Aoƍ DUHabsorbances measured at zero time of incubation for the test sample

and control, respectively; At and AtƍDUHthe absorbances measured in the test sample

and control, respectively, after incubation for 3 hr.

77

4.2.6 Superoxide dismutase assay


This assay was conducted with Sigma SOD assay kit [81]. SOD Assay Kit-WST

DOORZV YHU\ FRQYHQLHQW 62' DVVD\LQJ E\ XWLOL]LQJ 'RMLQGR¶V KLJKO\ ZDWHU-soluble

tetrazolium salt, WST-1 (2-(4-Iodophenyl)- 3-(4- nitrophenyl)-5-(2,4-disulfophenyl)-

2H-tetrazolium, monosodium salt) that produces a water-soluble formazan dye upon

reduction with a superoxide anion. The rate of reduction with oxygen is linearly

related to the xanthine oxidase (XO) activity, and is inhibited by SOD. Therefore, the

IC50 (50% inhibition activity of SOD or SOD-like materials) can be determined by a

colorimetric method. Since the absorbance at 450 nm is proportional to the amount of

superoxide anion, the SOD activity as an inhibition activity can be quantified by

measuring the decrease in the colour development at 450 nm.


Twenty microliters of sample was plated at different concentrations in a 96-well

microtiter plate. Then, 200 µl of WST solution and 20 µl of enzyme was added and

incubated for 20 mins at 37 oC. Absorbance was then recorded at 450nm using

Thermo Scientific Varioskan Flash microtiter plate reader, linked to a computer

equipped with (SkanIt Software 2.4.3) and SOD inhibition rate was determined. SOD

enzyme (Nacalai Tesque) was used as standard control.

78



The total phenolic content of the crude extract and fractions was determined with the

Folin±Ciocalteu reagent, following the modified method of Singleton and Rossi [82].

The Folin-Ciocalteu reagent is sensitive to reducing compounds including

polyphenols, thereby producing a blue colour upon reaction. This blue colour is

measured spectrophotometrically, thus determining the total phenolic content.


Briefly, 20 µl of test samples dissolved in methanol (1 mg/ ml) were added to 100 µl

of Folin±Ciocalteu reagent and 1.58 ml of deionised water. After allowing the mixture

to stand at room temperature for 5 min, 300 µl of 20% (w/v) sodium carbonate were

added. Reaction mixtures were further incubated at room temperature for 30 mins,

following which absorbance at 765 nm was read against a blank, using a Jenway 6305

UV±Vis spectrophotometer (Jenway Ltd., Essex, UK). The standard calibration curve

was plotted using gallic acid (50-250 µg/ml), from which total phenolic content was

expressed as gallic acid equivalents (mg/g extract).

79

4.2.8 Total flavonoid content determination


The total flavonoid content of the crude extract and fractions was determined with the

aluminium chloride colorimetric method of Froehlicher et al. [83]. The principle of

this assay is that aluminium chloride forms acid stable complexes with the C-4 keto

group and either the C-3 or C-5 hydroxyl group of flavones and flavonols. In addition,

aluminium chloride forms acid labile complexes with the orthodihydroxyl groups in

the A- or B- ring of flavonoids [84].


Briefly, 0.5 ml of test samples dissolved in methanol (1 mg/ ml) were added to 1.5 ml

of 2% methanolic solution of aluminium chloride in sealed tubes and kept in dark for

15 mins. Absorbance was then read at 430 nm using UV-vis spectrophotometer

against blank of methanolic aluminium chloride solution. The standard calibration

curve was plotted using quercetin (50-250 µg/ml), from which total flavonoid content

was expressed as quercetin equivalents (mg/g extract).

4.2.9 Statistical analysis

Concentration±response curves were calculated using the Prism software package

5.00 for Windows, GraphPad Software, San Diego California USA,

www.graphpad.com (GraphPad, San Diego, USA) and data were reported as mean

80

and SD values obtained from a minimum of three determinations. Non linear best fit

was plotted with SD and 95% confidence interval. All data were expressed as mean ±

standard deviation. SD was computed but values were not shown in the graphs via

this software. Data were analyzed using one way Anova followed by Tukey test using

GraphPad Prism5 software. A significant difference was considered at the level of P <

0.01.

4.3 Results

4.3.1 DPPH assay

In this test the ethanol extract of leaves and barks exhibited profound antioxidant

activities (Figure 4.5). The antioxidant activities were highest for ethanol extract of

barks (77.80±0.01 %) followed by the ethanol extract of leaves (65.80±0.01 %), the

hexane extract of leaves (38.90±0.02 %), the chloroform extract of barks (8.00±0.02

%), the hexane extract of barks (5.00±0.01 %) and the chloroform extract of leaves

(3.00±0.01 %) against standard ascorbic acid which was 97.90±0.00 % at the dose of

10 µg/ml (Table 4.1). The EC50 (µg/ml) for the ethanol extract of barks (2.33 µg/ml)

and leaves (2.93 µg/ml) were as good as ascorbic acid (1.88 µg/ml) as shown in

Figure 4.6 and Table 4.2.

81

Figure 4.5: DPPH test result with 96 well microtiter plate results for bark (left) and leaf

(right) ethanol extract.

4.3.2 ABTS assay

In this test the ethanol extract of leaves and barks exhibited good antioxidant activities

(Figure 4.7). The EC50 (µg/ml) for the ethanol extract of barks (0.93±0.01 µg/ml) and

leaves (2.28±0.01 µg/ml. The EC50 for ethanol extract of bark was significantly lower

than ascorbic acid (1.54±0.06 µg/ml) (Table 4.2).

4.3.3 FRAP assay

The FRAP values are represented in terms of concentration of sample that produced

same absorbance as 1 mM of Fe (II) was determined (y of sample filled in equation to

obtain x). The antioxidant activity was calculated as Ferrous Equivalents, the

concentration of samples which produced an absorbance value equal to that of 1 mM

FeSO4.

82

Table 4.1: DPPH radical scavenging activity at 10 µg/ml of extract

Extracts % DPPH radical scavenging

LH 38.90±0.02

LC 3.00±0.01

LE 65.80±0.01

BH 5.00±0.01

BC 8.00±0.02

BE 77.80±0.01

AA 97.90±0.00

LH-leaf hexane extract, LC- leaf chloroform extract, LE-leaf ethanol extract

BH- bark hexane extract, BC- bark chloroform extract, BE- bark ethanol extract

AA- ascorbic acid

83

0 2 4 6 8 10

0

20

40

60

80

100

AA

LH

LC

LE

BH

BC

BE

Quercetin

Trolox

concentration(Pg/ml)

% D

PP

H

Figure 4.6: DPPH scavenging activity (%) of Canarium patentinervium Miq.

84

0 20 40 60

0

20

40

60

80

100

LH

LC

LE

BH

BC

BE

Trolox

QuercetinAscorbic acid


% A

BT

S d

ecol

ouri

satio

n

Figure 4.7: ABTS scavenging activity (%) of Canarium patentinervium Miq.

85

Linear regression curve for all samples and positive controls are shown in Figure 4.9-

4.17. From the standard graph of ferrous sulphate (Figure 4.8), the values for six

extracts were determined using the standard formula (Table 4.2). The FRAP activity

was calculated from regression equation of ferrous sulphate calibration curve

(y=0.0105x+0.0136, r2- 0.9817). The ethanol extract of bark showed significant

FRAP activity of 67.00±0.32 µg/ml compared to quercetin and ascorbic acid. The

ethanol extract of the leaves showed significant FRAP activity of 200.00±0.07 µg/ml

compared to ascorbic acid. The poorest activity was shown by hexane extract of barks

that correlates with the low r2 in its regression equation (Figure 4.12).

4.3.4 ȕ-carotene bleaching assay

The ȕ-carotene decolourisation (Figure 4.18) was most prominent in ethanol extract

of leaves and barks with EC50 (µg/ml) of 6.04±0.02 µg/ml and 7.04±0.04 µg/ml

respectively (Table 4.2)

4.3.5 Superoxide dismutase assay

The ethanol extract of the leaves shows highest SOD-like activity with IC50 of

ȝJPO7DEOH.


The total phenolics in the extracts was determined with the Folin-Ciocalteu assay,

calculated from regression equation of calibration curve of gallic acid

86

(y=0.1762x+0.0047, r2- 0.9994) (Figure 4.19) and is expressed as gallic acid

equivalents, GAE (mg/g). The highest phenolic content was in ethanol extract of

leaves followed by barks (Table 4.2).

4.3.7 Total flavonoid content determination

The total flavonoids in the extracts was determined with the aluminium chloride

assay, calculated from regression equation of calibration curve (y=0.09330x+0.0023,

r2-0.9992) (Figure 4.20) of quercetin and is expressed as quercetin equivalent, (mg/g).

The highest flavonoid content was in ethanol extract of leaves followed by barks

(Table 4.2).

4.3.8 The antioxidant values, total phenolic and flavonoid content and their

correlations

The total antioxidant activity investigated with the relevant total phenolic and

flavonoid content are summarized in table 4.3.

87

Figure 4.8: Fe (II) calibration curve in FRAP assay.

y = 0.0105x + 0.0136 r² = 0.9817

0

0.1

0.2

0.3

0.4

0.5

0.6

0 10 20 30 40 50 60

Ab

sorb

ance

, 59

3n

m

Concentration µM

FRAP: Fe(II) calibration curve

88

Figure 4.9: Leaf hexane (LH) extract calibration curve in FRAP assay.

y = 0.0036x + 0.2681 r² = 0.9576

0.0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40 50 60

Ab

sorb

ance

, 59

3 n

m

Concentration µg/ml

Frap LH

89

Figure 4.10: Leaf chloroform (LC) extract calibration curve in FRAP assay.

y = 0.0077x + 0.2794 r² = 0.9857

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30 40 50 60

Ab

sorb

ance

,59

3n

m


FRAP: LC calibration curve

90

Figure 4.11: Leaf ethanol (LE) extract calibration curve in FRAP assay.

y = 0.0525x + 0.0136 r² = 0.9817

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6 8 10 12 14

Ab

sorb

ance

,59

3n

m


FRAP: LE calibration curve

91

Figure 4.12: Bark hexane (BH) extract calibration curve in FRAP assay.

y = 0.0029x + 0.2733 r² = 0.6982

0

0.1

0.2

0.3

0.4

0.5

0 10 20 30 40 50 60

Ab

sorb

ance

,59

3n

m


FRAP: BH calibration curve

92

Figure 4.13: Bark chloroform (BC) extract calibration curve in FRAP assay.

y = 0.0082x + 0.3288 r² = 0.9894

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30 40 50 60

Ab

sorb

ance

,59

3n

m


FRAP: BC calibration curve

93

Figure 4.14: Bark ethanol (BE) extract calibration curve in FRAP assay.

y = 0.1652x + 0.3254 r² = 0.9989

0.0

0.5

1.0

1.5

2.0

2.5

0 2 4 6 8 10 12 14

Ab

sorb

ance

,59

3n

m


FRAP: BE calibration curve

94

Figure 4.15: Ascorbic acid (AA) calibration curve in FRAP assay.

y = 0.0301x + 0.0831 r² = 0.9799

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 10 20 30 40 50 60

Ab

sorb

ance

,59

3n

m


FRAP: AA calibration curve

95

Figure 4.16: Trolox (TRO) calibration curve in FRAP assay.

y = 0.1193x + 0.2815 r² = 0.9904

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

0 2 4 6 8 10 12 14

Ab

sorb

ance

,59

3n

m


FRAP: TRO calibration curve

96

Figure 4.17: Quercetin (QC) calibration curve in FRAP assay.

y = 0.3053x + 0.4857 r² = 0.9646

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0 2 4 6 8 10 12 14

Ab

sorb

ance

,59

3n

m


FRAP: QC calibration curve

97

0 20 40 60

0

20

40

60

80

100

LH

LC

LE

BH

BC

BE

TROLOX

QUERCETIN


%E -

caro

tene

dec

olou

risat

ion

Figure 4.18ȕ-carotene decolourisation (%) of Canarium patentinervium Miq.

98

Gallic Acid calibration curve

y=0.1762x+0.0047, r2=0.9994

0 2 4 6

0.0

0.2

0.4

0.6

0.8

1.0


Abso

rban

ce (O

765

nm)

Figure 4.19: Gallic acid calibration curve in Folin-Ciocalteu assay.

99

Quercetin calibration curve

y=0.009330x-0.002314, r2=0.9992

20 40 60

-0.1

0.0

0.1

0.2

0.3

0.4

0.5

Concentration (Pg/ml)

Absorb

ance (O

430 n

m)

Figure 4.20: Quercetin calibration curve in total flavonoid content determination.

100

Table 4.2: Antioxidant values of Canarium patentinervium Miq.

Extracts

Total phenolic content, Gallic acid equivalent

(mg/g)

Total flavonoid content, Quercetin equivalent (mg/g)

ABTS assay, EC50 ȝJPO

DPPH assay, EC50 ȝJPO

FRAP assay, FRAP value

ȝJPO

ȕ-carotene bleaching

assay, EC50

ȝJPO

Superoxide dismutase

assay IC50ȝJPO

LH 6.61±0.01 18.99±0.02 27.30±0.01 17.45±0.03 2845.00±0.15 9.39±0.05 80.54±0.03

LC 14.28±0.03 12.57±0.04 31.10±0.02 134.00±0.04 1329.00±0.18 21.81±0.04 26.84±0.03

LE 204.97±0.05 125.32±0.03 2.28±0.01 2.93±0.00B 200.00±0.07 6.04±0.02 3.05±0.01

BH 5.19±0.01 1.55±0.02 521.00±0.01 1857.00±0.01 3531.00±0.14 125.00±0.08 238.00±0.04

BC 13.99±0.03 12.14±0.02 34.90±0.02 87.80±0.05 1242.00±0.19 50.40±0.03 20.13±0.02

BE 100.26±0.01 24.57±0.02 0.93±0.01A 2.33±0.02 67.00±0.32 7.04±0.04 8.51±0.07

AA NA NA 1.54±0.06 1.88±0.01 347.00±0.23 NA NA

QC NA NA 0.88±0.03A 2.88±0.02B 86.00±0.24 1.64±0.04C NA

TRO NA NA 0.68±0.02 4.77±0.04 33.00±0.54 1.65±0.03C NA

SOD NA NA NA NA NA NA 1.59±0.04

LH: leaf hexane extract, LC: leaf chloroform extract, LE: leaf ethanol extract, BH: bark hexane extract, BC: bark chloroform extract, BE: bark ethanol extract, AA: ascorbic acid, QC:quercetin, TRO: trolox and SOD: superoxide dismutase Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same capital letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

101

Table 4.3: The mean r2 values for correlation between total phenolics contents, total flavonoid contents and antioxidant activities (EC50)

ABTS DPPH FRAP BC

Total phenolic contents 0.1410 0.1285 0.5246 0.2287

Total flavonoid contents 0.1388 0.1319 0.3179 0.2146

ABTS na 0.9977*** 0.5398 0.9008**

DPPH 0.9977*** na 0.5029 0.8977**

FRAP 0.5398 0.5029 na 0.4770

BC 0.9008** 0.8977** 0.4770 na

** significant with p<0.01

*** significant with p<0.0001, na- not applicable

102

4.4 Discussion

In vitro antioxidant capacity can be determined by hydrogen atom transfer (HAT)

method and single electron transfer (SET) method [56]. HAT based methods measure

the ability of an antioxidant to scavenge free radical by hydrogen donation to form a

stable compound. SET based methods detect the ability of the antioxidant to transfer

one electron to reduce compound including metals, carbonyls and radicals [70][85]. ȕ-

carotene bleaching assay involves HAT method, FRAP assay involves SET method

while DPPH and ABTS assay involves both method predominantly via SET method

[55][57].

FRAP is the ferric reducing power of antioxidants by the reduction of the ferric ions

to the ferrous ions, which form a blue colored ferrous-tripyridyltriazine complex

(ferric TPTZ) which is detected at 593 nm. Deeper blue colour indicates higher

antioxidant potential [86]. The samples absorbance equivalent to 1 mM FeSO4 was

calculated from equation of linearity of individual sample. Total FRAP value was

determined from the absorbance obtained from samples above using the standard Fe

(II) calibration curve equation (y=0.0105x+0.0136, r2=0.9817). Ethanol extract of

barks displayed significant (p< 0.01) FRAP value ( ȝJPO compared to

ascorbic acid (ȝJPODQGTXHUFHWLQȝJPO

,Q WKH ȕ-carotene/linoleic model, linoleic acid reacts with ROS and O2 to form an

XQVWDEOHSHUR[\UDGLFDOȕ-carotene being an antioxidant will react with this radical to

form stable epoxide causing the bleaching of yellow solution. Competition reaction

occurs with the presence of another antioxidant (sample) to react with the peroxy

103

radical resulting in slower bleaching of solution detected at 470 nm

spectrophotometrically [87]. Ethanol extract of leaves and barks displayed best EC50

value of ȝJPO DQG ȝJPO UHVSHFWLYHO\ Our results are in

agreement to the previous reports which pointed out that ascorbic acid did not show

its antioxidant activity under similar assay [88] Such phenomenon was termed as

³SRODU SDUDGR[´ZKHUHE\ SRODU DQWLR[LGDQWV UHPDLQLQJ LQ WKH DTXHRXV SKDVH RI WKH

emulsion are more diluted in lipid phase and thus, they are less effective [89].

ABTS assay involves the reduction of the blue-green 2,2-azino-bis(3-

thylbenzothiazoline-6- VXOIRQDWH UDGLFDO FDWLRQ $%76ǜ E\ DQWLR[LGDQWV WR LWV

original colourless ABTS form. Greater discolourisation results in lower absorbance

at 734 nm indicating higher antioxidant capacity [75][76]. Ethanol extract of the bark

displayed significant (p<0.01) EC50 ( ȝJPO) compared to ascorbic acid

(ȝJPO).

The DPPH radical has a deep purple colour and absorbs strongly at a wavelength of

550 nm, whereas the yellowish reduction product DPPH2 does not. In DPPH assay

(another SET method), ethanol extract of leaves and barks displayed significantly

lower EC50 value of ȝJPODQGȝJPOUHVSHFWLYHO\FRPSDUHGWR

trolox (EC50 = ȝJPO[90]. The radical scavenging potential against DPPH

organic radical directly depends on the number of hydroxyl groups present in ring B

of flavonoids, with an increase in the number of hydroxyl groups resulting in an

increase in radical scavenging activity [91]. Phenols, amino and thiophenol groups are

commonly known to be the active groups for DPPH scavenging. The mechanism by

which DPPH is scavenged, aids in elucidating the structure-activity relationship

104

(SAR) of the antioxidant and in doing so, may be beneficial in the rational design of

novel flavonoid-derived antioxidants with improved pharmacological profiles [92].

The DPPH and ABTS assays have the same mechanism of action, but, in most cases,

the results obtained from the ABTS assay are higher than those from DPPH assay. It

has been documented that results reported for the ABTS assay do not only take into

account the activity of the parent compound, but also the contribution of reaction

products and other individual compounds on the activity, which is not the case in the

DPPH assay [93][94].

SOD which catalyses the dismutation of the superoxide anion (O2-) into hydrogen

peroxide and molecular oxygen, is one of the most important anti-oxidative enzymes.

Preventive antioxidants, such as SOD are described either as preventing introduction

of initiating radicals or as inhibiting the rate which new chains are set up. The

analytical methods for determination of O2- scavenging capacity employ enzymatic or

nonenzymatic O2- generation systems. In this study the enzymatic system that uses

xanthine oxidase (XOD) to generate O2- radicals was used [57]. XOD was used for

formation of O2- that is the reduced to oxygen by SOD-like activities in samples. The

WST-1 is used as a probe that undergoes competitive reaction with SOD-like samples

to reduce O2- to oxygen forming a formazan coloured dye. Thus higher rate of SOD

activity results in less dye formation and less absorbance. The ethanol extract of the

leaves shows highest SOD-like activity with IC50 RIȝJPO

In the above experiments, the ethanol extract of leaves and barks displayed superior

antioxidant capacities. The EC50 values of the samples were consistently low in SET

105

methods (ABTS, DPPH and FRAP) superior to standard as opposed to HAT method

ȕ-carotene bleaching) assay. The present study demonstrates that Canarium

patentinervium Miq. is a potent source of antioxidants that exhibits its antioxidant

activity predominantly via the SET method. Several reports emphasized on the fact

that there is a positive relationship between total phenolic contents and antioxidant

activity [95][96]. Our study demonstrated the existence of a low positive correlation

between the total phenolic contents and the FRAP values (y = -0.0125x + 2.2564, r2 =

0.5246) and total phenolic contents and ȕ-carotene bleaching values (y = -0.2747x +

52.42, r2 = 0.2287), respectively. Therefore, one could draw an inference that non

phenolic by itself or in synergy with the phenolics and flavonoids impart Canarium

patentinervium Miq its antioxidant properties.

4.5 Conclusion

The present study demonstrates that Canarium patentinervium Miq. is a potent

source of antioxidants that exhibits its antioxidant activity predominantly via the SET

method. Pronounced radical scavenging activity has been reported in plants with

phenolic moieties, the presence of which is common in natural antioxidants. These

phenolic moieties include substances such as tannins, flavonoids, phenolic acids,

tocopherol, and cathecheses. Tannins are, at least in part, responsible for the free

radical scavenging activities working synergistically with other antioxidant substances

[86].

106

CHAPTER 5

ANTI-INFLAMMATORY ACTIVITY

5.1 Introduction

5.1.1 Inflammation and arachidonic system

Over the last 10 years, a significant body of evidence has emerged indicating that

chemically diverse classes of naturally-occurring substances derived from higher

plants are of potential interest for therapeutic interventions in several inflammatory

diseases [97]. Inflammation response is a critical protective reaction to irritation,

injury, or infection, characterised by redness, heat, swelling, loss of function and pain.

Redness and heat result from an increase in blood flow, swelling is associated with

increased vascular permeability, and pain is the consequence of activation and

sensitisation of primary afferent nerve fibres. In normal conditions, these changes in

inflamed tissue serve to isolate the effects of the insult and thereby limit the threat to

the organism [97].

In recent years, roles have been identified for several inflammatory cells and for a

large number of inflammatory mediators in important pathologies not previously

OLQNHG WR LQIODPPDWLRQ VXFK DV $O]KHLPHU¶V GLVHDVH DQG FDUGLRYDVFXODU GLVRUGHUV

including atherosclerosis, as well as cancer [98]. These findings have significantly

increased the importance of understanding the cellular and molecular mechanisms

forming the basis of the inflammatory process and in identifying new targets for the

107

development of innovative and safe therapeutic strategies for the management of

inflammatory diseases. Most plant-derived secondary metabolites are known to

interfere directly or indirectly with the following molecules and/or mechanisms

[98][99]:

1. various inflammatory mediators (eg arachidonic acid metabolites, peptides,

cytokines, excitatory amino acids etc).

2. the production and/or action of second messengers ( such as cGMP, cAMP,

various protein kinases, and calcium among others).

3. the expression of transcription factors such as AP-1, NF-Ƹ% DQG SURWR-

oncogenes (c-jun, c-fos, and c-myc),

4. the expression of key pro-inflammatory molecules such as inducible NO

synthase (iNOS), cyclooxygenase (COX- F\WRNLQHV ,/ ȕ 71)-Į HWF

neuropeptidases and proteases.

In this study we reviewed and investigated the contribution of naturally occurring

substances from higher plants acting on the arachidonic acid pathway. Inflammation

in injured cells is both initiated and maintained by the overproduction of

prostaglandins and leukotrienes, which are produced by separate enzymatic pathways,

viz. the cyclo-oxygenase (COX) and lipoxygenase (LOX) pathways, respectively.

Both the prostaglandins as well as the leukotrienes are biosynthesised on demand

from arachidonic acid, which is a 20-carbon fatty acid, derived from the breakdown of

cell membrane phospholipids by any number of phospholipase A2 (PLA2) isoforms

[100] (Figure 5.1).

108

.

Other HPETEs

lipoxygenases

HETEs

Prostaglandin G2 (PGG2) 5-HPETE 5-HETE

Prostaglandin H2 (PGH2)

Leukotriene A4 (LTA4) LTB4

Prostacyclin Thromboxane A2

(PGI2) (TXA2) Leukotriene C4 (LTC4)

Leukotriene D4 (LTD4)

Leukotriene E4 (LTE4)

PGD2 PGE2 Lipoxin A4 Lipoxin B4

(LXA4) (LXB4)

Figure 5.1: Arachidonic acid metabolites and inflammation [101].

Cell membrane phospholipids

ARACHIDONIC ACID

Phospholipases

Cyclooxygenase 5-Lipoxygenase

Causes vasodilation,

inhibits platlet

aggregation

Causes vasocontriction,

promotes platlet

aggregation

Vasodilation Increased vascular

permeability

Chemotaxis

Vasoconstriction Bronchospasm Increrased vascular permeability Inhibit neutrophil

adhesion and chemotaxis

12-

109

Arachidonic acid is normally stored in membrane-bound phospholipids and released

by the action of phospholipases. Enzymatic conversion of released arachidonic acid

into biologically active derivatives proceeds through one of several routes [99].

Cyclooxygenase converts arachidonic acid to unstable cyclic endoperoxides from

which prostaglandins, prostacyclin and thromboxanes are derived. Formation of the

leukotrienes from arachidonic acid is initiated by the action of 5-lipoxygenase

producing leukotriene A4. Hydrolysis of leukotriene A4, or the incorporation of

glutathione results in the formation of leukotriene B4 and C4, respectively [102]. In

addition, 12- and 15-lipoxygenase can catalyse arachidonic acid conversion and

lipoxins A and B are amongst the possible products. Many of these metabolites of

arachidonic acid feature prominently in the development of inflammation.

Prostaglandin E2 and prostacyclin are potent vasodilators, while leukotriene D4 causes

cellular adhesion, chemotaxis of neutrophils and degranulation. Leukotrienes C4, D4

and E4 contribute to inflammation by increasing vascular permeability. Leukotrienes

are also believed to play an important pathophysiological role in allergic broncho-

constriction of asthma [103].

Through pharmacological intervention in the arachidonic acid cascade various anti-

inflammatory agents have been developed. These include aspirin-like drugs, which

inhibit cyclo-oxygenase. Corticosteroids appear to indirectly inhibit phospholipases

thus preventing release of arachidonic acid. Future progress in this field is likely to

produce drugs which antagonise arachidonic acid derivatives or inhibit the enzymes

involved in their synthesis with greater specificity [99][103].

110

5.1.2 The lipoxygenase system

5-LOX presents either in the cytosol or the nucleus of a resting cell (depending on the

cell), as a soluble enzyme. Upon cellular stimulation, 5-LOX and cytosolic

phospholipase A2 (cPLA2) co-migrate to the nucleus. It is here that cPLA2 liberates

arachidonic acid from the membrane phospholipids [104] (Figure 5.2). Arachadonic

acid is the main substrate for the lipoxygenase enzyme, and is presented to 5-LOX for

metabolism by the 5-lipoxygenase activating protein (FLAP). The metabolism of

arachidonic acid produces leukotrienes and lipoxins via the LOX pathway [105]. 5-

LOX, catalysing the oxidation of arachidonic acid, produces 5(S)-

hydroxyperoxyeicosatetraenoic acid (5-HPETE), a hydroperoxide intermediate, which

undergoes dehydration, resulting in the formation of leukotriene A4 (LTA4).

Enzymatic hydrolysis of LTA4, as well as conjugation with other substances, leads to

the formation of inflammatory mediators [106]. These inflammatory mediators are

responsible for the powerful chemo-attractive effects on the eosinophils, neutrophils

and macrophages, as well as the increased release of pro-inflammatory cytokines by

macrophages and lymphocytes.

Two other pathways in the LOX system, 15-LOX and 12-LOX, are responsible for

the production of lipoxins, the latter having the potential to counteract the pro-

inflammatory effects of the leukotrienes. 5-Lipoxygenase is inhibited by quinones,

hydroxyquinones, and a variety of phenolic compounds, including certain flavonoids

such as, quercetin, kaempferol, morin, myricetin and cirsiliol [107]. At the onset of

the inflammatory process, arachidonic acid is converted to eicosanoids and

leukotriene B4 (LTB4) by LOX [104], which is coupled with the production of

111

prostaglandins and thromboxanes by cyclooxygenase (COX). The leukotrienes

present at the onset of inflammation are also responsible for the maintenance thereof.

Leukotrienes have been identified as mediators of a number of inflammatory and

allergic reactions [103]..

These include rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis,

psoriasis, chronic urticaria, asthma [108] and allergic rhinitis [109][103]. The 5-LOX

pathway has also recently been linked to the development of atherosclerosis,

osteoporosis and certain types of cancers [105]. As a result of the pathophysiological

implications of 5-LOX products and the potential benefits of anti-leukotriene therapy,

different strategies have been employed (targeting PLA2, 5-LOX, FLAP, LTA4

hydrolase and leukotriene C4 (LTC4) synthase with 5-LOX being the ideal and most

promising target [105].

5.1.3 The cyclooxygenase system

Cyclooxygenase (COX) is a key enzyme that catalyzes the transformation of

arachidonic acid to several biologically active eicosanoids, such as prostaglandins

(PGs), thromboxane (e.g., TxA2), and prostacyclin (e.g., PGI2). This enzyme actually

catalyzes two major reactions, as shown in Figure 5.3. Arachidonic acid, the most

important physiological precursor of eicosanoids biosynthesis, can be released from

the membrane phospholipid by activation of phospholipase A2 (PLA2) and/or

phospholipase C (PC). Cyclooxygenase then catalyzes the addition of molecular

oxygen at the C-11 of arachidonic acid to form the 11-peroxy intermediate, following

112

by rearrangement to a cyclic endoperoxide and introduction of a second molecule of

oxygen at C-15 to form prostaglandin G2 (PGG2).

Ligand

Receptor

Figure 5.2: The translocation of 5-lipoxygenase and cytosolic phospholipase A2, upon

cellular stimulation, to the nuclear membrane, followed by substantial generation of

leukotrienes [105]. 5-LOX = 5-lipoxygenase, cPLA2 = cytosolic phospholipase A2, FLAP =

5-lipoxygenase activating protein, PKA = protein kinase A, cAMP = cyclic adenosine

monophosphate, MK2 = kinases, ERK1,2 = extracellular signal-regulated kinase 1,2.

Cell Stimulation

5-LOX

?

Phospholipase C

5-LOX

MAP Kinases ERK 1,2

+ Ca 2+

+ DAG

?

FLAP

cPL

Nucleu

s

Leukotrienes

SH3- Interactions

Antiinflammatory Signal (cAMP)

CELL

p 38 MK2

113

The hydroperoxidase activity reduces PGG2 to generate PGH2 which is the immediate

precursor for production of eicosanoids, such as PGD2, PGE2, PGF2a, PGI2, and TxA2,

through corresponding synthase activities. These arachidonic acid metabolites derived

from PGH2 are important mediators of inflammatory responses, immunological

effects, and tumour development [100].

Cyclooxygenase (COX) exist as three isoforms: cyclooxygenase-1 (COX-1),

expressed in most cells constitutively, inducible cyclooxygenase-2 (COX-2), triggered

by pro-inflammatory stimuli, and COX-3, present mainly in the cerebral cortex and

human heart [110][111] . COX-1 is a constitutive form of the enzyme that has been

linked to the production of physiologically important prostaglandins that may play a

role in homeostasis (gastric, renal, etc). COX-2 exists in a constitutive form in kidney

and brain and is inducible by cytokines and growth factors. Induction of the COX-2 is

linked to inflammatory cell types and tissues and COX-2 is believed to be the target

for the anti-inflammatory actions of NSAIDs. Thus there is an on-going effort to

identify compounds that will inhibit COX-2 in preference to COX-1 since such an

agent may be safer and perhaps more efficacious [112].

Despite of the undesired side effects such as increased cardiovascular risk of COX-2-

selective inhibitors, much research spanning many years has been focused on finding

COX-2 selective inhibitors, owing to the remarkable reduction of adverse

gastrointestinal and renal effects associated with conventional non-steroidal anti-

inflammatory drugs (NSAIDs) as well as to their potential therapeutic benefits in

several diseases, including certain types of cancer [113]. Thus, it is of value to study

cyclooxygenase inhibitors.

114

Phospholipase A2

Arachidonic acid

Constitutive & Constitutive Inducible

PGG2

Hydroperoxidase

PGH2

Prostanoids

Receptors

SOME BIOLOGICAL EFFECTS

Figure 5.3: Cyclooxygenase and arachidonic acid metabolism.

Membrane phospholipids

COX-2 COX-1

Cell specific isomerases

PGI2 TXA2 PGE2 PGD2 PGFĮ

Prostacylin synthase PGE Synthase 3*)Į6\QWKDVH

Thromboxane Synthase

PGD Synthase

IP 73Į73ȕ EP1, EP2,

EP3, EP4

DP1, DP2 )3Į)3ȕ

Vasodilator Antiaggregant

Vasoconstrictor Proaggregant

Vasodilator Neuroprotective Neurodestructive

Vasodilator Anti- inflammatory Proapoptotic

Vasodilator Antiaggregant

115

5.1.4 Dual inhibitors

Conventional pharmacological management of inflammatory disease like

osteoarthritis involves treatment with non-steroidal anti-inflammatory drugs

(NSAIDs) or selective COX-2 inhibitors that block the formation of PGs without

modulating 5-LOX enzyme activity. Inhibition of one or both of the COX enzymes

PD\³VKXQW´$$PHWDEROLVPGRZQWKH-LOX pathway, which can aggravate toxicity

associated with the lack of PGs and excess production of LTs. For example, NSAID-

induced gastric ulcers have been shown to have high concentrations of LTB4 in their

walls, which attracts leukocytes to the stomach and may contribute to ulceration

[114][115].

Recently, reports have appeared regarding so-FDOOHG ³GXDO LQKLELWRUV´ DJHQWV WKDW

inhibit not only COX-1 and COX-2, but also 5- LOX [116][117][118][119]. These

agents with anti-oxidative properties may be particularly effective for managing the

metabolic processes underlying inflammatory conditions and reducing both gastric

and cardiovascular side effects by balancing AA metabolism in the body. This dual

inhibition activity will be investigated for this plant.

5.1.5 Anti-inflammatory potential of Canarium L.

The essential oil of Canarium schweinfurthii Engl. collected from the region of

Cameroon inhibited the enzymatic activity of lipoxygenase with an IC50 value of 62.6

ppm [19]. However in a separate study of the same species collected from central

African region did not show any activity in the cotton pellet induced granuloma

116

formation experiment [13]. This may suggest possible evidence of variety between

secondary metabolites constituents according to regions. The essential oil of

Canarium schweinfurthii Engl. at a dose of 1, 2 and 3 mg/ kg i.p. displayed potent

analgesic effect in the acetic acid-induced writhing and hot plate experiments [13].


5.2.1 Anti-inflammatory determination assays

The anti-inflammatory activity of the plant extracts was determined using the in vitro

5- LOX assay. This assay measures the inhibitory activity against the 5-LOX enzyme,

which is a key enzyme in the metabolism of arachidonic acid that is responsible for

the formation of leukotrienes (which play a pivotal role in the pathophysiology of

chronic inflammatory and allergic diseases) as first determined by Sircar et al. [120]

and later modified by Evans [121].

There are three ways to measure COX activity, one is to measure the final product of

the reaction, PGE2; another is to measure directly the oxygen uptake that occurs

during the first step of the reaction using the oxygen sensor and third is to measure the

second reaction (peroxidase function) spectrophotometrically [112]. The peroxidase

co-substrate oxidation assay can detect cyclooxygenase inhibitors which may directly

interfere with enzyme activity or scavenge the free radical intermediates to block the

reaction process. However, the oxygen consumption assay is mainly reflective of

inhibitors which block the interaction of enzyme and arachidonic acid. We currently

117

describe a peroxidase co-substrate oxidation assay system adapted to a 96-well plate

format. This requires less enzyme and reaction material and yields savings in time and

money. The peroxidase co-substrate oxidation assay is useful as a pre-screening

method [100][112].

5.2.2 The 5-LOX inhibition assay


The 5-LOX assay was conducted according to the method described by Baylac &

Racine [122] with some modifications. Lipoxygenases (LOX) are dioxygenases that

catalyse the addition of molecular oxygen to polyunsaturated fatty acids containing a

1,4-pentadiene group. The 5-LOX enzyme thus converts its substrate, arachidonic

acid, to the conjugated diene product 5-hydroxy- 6,8,11,1-eicosatetraenoic acid (5-S-

HETE), which, in turn, is converted to LTA4, and then to LTB4, by LTA4 hydrolase.

For the purposes of this experiment, linoleic acid was used as the substrate, as it

shares a high degree of structural similarity to arachidonic acid (containing the 1,4-

pentadiene group in question), it is far easier to handle as well as having a stronger

affinity for the 5-LOX enzyme resulting in greater UV absorbance readings [123].

The experiment specifically determines increases in absorbance at 234 nm as a result

of the formation of conjugate double bonds in linoleic acid hydroperoxide (from a

1,4-diene to a 1,3-diene), as used in the biochemical evaluation of the LOX pathway

of soybean plants submitted to wounding [124].

118

5.2.2.2 Protocol

5-LOX enzyme (human recombinant from Calbiochem) was used. Ice-cold buffer

(potassium phosphate) at 4 oC was mixed with 100 U of the thawed enzyme. Twenty

microliters of samples dissolved in DMSO was plated out in triplicate in a 96-well

microtiter plate at different concentrations, followed by 160 µl of 0.1 M potassium

phosphate buffer (pH 6.3) maintained at 25 oC and 20 µl of enzyme solution. Mixture

was agitated and 10 µl of linoleic acid was added and incubated for 10 mins at 25 o C.

Absorbance was recorded at 234 nm using Thermo Scientific Varioskan Flash

microtiter plate reader, linked to a computer equipped with (SkanIt Software 2.4.3). 5-

Lipoxygenase is known to catalyse oxidation of unsaturated fatty acids containing 1-4

diene and the modification of linoleic acid (1-4-diene into 1- 3-diene) can be detected

at 234 nm. Percentage inhibition of enzyme was determined by comparison of rates of

reaction of samples relative to blank sample using the formula (E-S)/E x 100, where E

is the activity of enzyme without test sample and S is the activity of enzyme with test

sample. The experiments were done in triplicate. Nordihydroguaiaretic acid (NDGA)

was used as positive control.

5.2.3 The peroxidase endpoint assay for COX-1 and COX-2


The COX-1 and COX-2 peroxidase endpoint assay was conducted according to

method described by Gierse et al. [112]. The peroxidase endpoint assay system is

based on sequential reactions of the cyclooxygenase (generating PGG2) and the

119

peroxidase (converting PGG2 to PGH2) activities. Its success relies on the peroxidase

co-substrate being transformed to a comparatively stable chromophore in a

predictable stoichiometric relationship to the reduction of PGG2. N,N,N¶,N¶-

tetramethyl-p-phenylenediamine (TMPD) is a convenient co-substrate for use in the

assay.

5.2.3.2 Protocol

COX-1 and COX-2 enzyme from Calbiochem was used. Twenty microliters of

sample dissolved in DMSO was plated in triplicates at different concentrations in a

96-well microtiter plate followed by 20 µl of enzyme solution. Then, 160 µl of

endpoint assay mix was added and incubated for 10 mins at room temperature.

Absorbance was recorded at 611 nm using Thermo Scientific Varioskan Flash


Percentage inhibition of enzyme was determined by comparison of rates of reaction of

samples relative to blank sample using the formula (E-S)/E x 100, where E is the

activity of enzyme without test sample and S is the activity of enzyme with test

sample. Indomethacin was used as positive control.





and SD values obtained from a minimum of three determinations. Non linear best fit

120



this software. Data were analyzed using one way Anova followed by Tukey test

using GraphPad Prism5 software. A significant difference was considered at the level

of P < 0.01.

5.3 Results

5.3.1 The 5-LOX inhibition assay

In the 5-LOX assay, chloroform extract of the barks displayed potent enzyme

inhibition (IC50=29.53±0.03 µg/ml) which was close to nordihydroguaiaretic acid

(IC50=29.19±0.02 µg/ml). Ethanol extract of leaves and barks follow through with

IC50=49.66±0.02 µg/ml and IC50=59.06±0.07 µg/ml respectively. Thus the potency of

activity against 5-LOX was BC > LE > BE (Table 5.1.). According to the 5-LOX

enzyme inhibition activity measurement guide by Kamatou et al. [94], [IC50 <30

µg/ml: good activity; 30 < IC50 <80 µg/ml : moderate activity; IC50 >80 µg/ml : poor

activity] chloroform extract of barks of Canarium patentinervium Miq displays good

5-LOX enzyme inhibition activity followed by moderate activity by ethanol extract of

leaves and barks.

121

5.3.2 The peroxidase endpoint assay for COX-1 and COX-2

In the COX assay, only ethanol extract of leaves and barks exhibited IC50 below 100

µg/ml. Ethanol extract of leaves had superior COX-1 inhibition (IC50 = 0.60±0.01

µg/ml) compared to COX-2 inhibition (IC50 = 1.07±0.01 µg/ml) (Figure 5.5) whereas

the barks had superior COX-2 inhibition (IC50 = 9.39±0.03 µg/ml) as opposed to

COX-1 (IC50 = 11.41±0.03 µg/ml) (Figure 5.4) .

5.4 Discussion

These assay measures the inhibitory activity against 5-LOX and COX enzymes. At

the onset of the inflammatory process, arachidonic acid is converted to eicosanoids

and leukotriene B4 (LTB4) by LOX [104], which is coupled with the production of

prostaglandins and thromboxanes by cyclo-oxygenase (COX). 5-LOX is the key

enzyme in the metabolism of arachidonic acid that is responsible for the formation of

leukotrienes which play a pivotal role in the pathophysiology of chronic inflammatory

and allergic diseases. Lipoxygenase catalysing the oxidation of arachidonic acid,

produces 5(S) - hydroxyperoxyeicosatetraenoic acid (5-HPETE), a hydroperoxide

intermediate, which undergoes dehydration, resulting in the formation of leukotriene

A4 (LTA4). Enzymatic hydrolysis of LTA4, as well as conjugation with other

substances, leads to the formation of inflammatory mediators [106]. These

inflammatory mediators are responsible for the powerful chemo-attractive effects on

122

0 20 40 60 80 100

0

20

40

60

80

100

COX-1

COX-2


% inhib

itio

n o

f C

OX

Figure 5.4: COX inhibition by BE.

123

0 2 4 6

0

20

40

60

80

100

COX-1

COX-2

% in

hibi

tion

of C

OX


Figure 5.5: COX inhibition by LE.

124

Table 5.1.Anti-inflammatory values of Canarium patentinervium Miq.

Extracts Anti-inflammatory assay 5-LOX, IC50

ȝJPO

COX-1 inhibition assay IC50ȝJPO


COX-1/COX-2 ratio

LH 206.00±0.02 >100 >100 NA

LC 104.69±0.04 >100 >100 NA

LE 49.66±0.02 0.60±0.01 1.07±0.01 0.56

BH 110.07±0.04 >100 >100 NA

BC 29.53±0.03 >100 >100 NA

BE 59.06±0.07 11.41±0.03 9.39±0.03 1.22

NDGA 29.19±0.02 NA NA NA

INDO NA 0.29±0.04 0.26±0.03 1.11

LH: leaf hexane extract, LC: leaf chloroform extract, LE: leaf ethanol extract, BH: bark hexane extract, BC: bark chloroform extract, BE: bark ethanol extract, AA: ascorbic acid, QC: quercetin, TRO: trolox and INDO: indomethacin. Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same capital letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

125

the eosinophils, neutrophils and macrophages, as well as the increased release of pro-

inflammatory cytokines by macrophages and lymphocytes [104].

Other inflammatory mediators such as histamine and immunoglobulin E, cause

spasms in the smooth muscle of bronchi and blood vessels, playing an eminent role in

asthmatic attacks. As leukotrienes thus play a major role in the pathophysiology of

chronic inflammatory diseases, it has been suggested that 5-LOX inhibitors may thus

be useful in the treatment of various conditions [125]. The 5-LOX pathway has also

been implicated in cardiovascular disease, including atherosclerosis, stroke,

myocardial infarction and the weakening of large artery walls and the formation of

aneurysms [126][105]. The presence of the 5-LOX pathway, the production of

leukotrienes and presence of the enzymes concerned, as well as leukotriene receptors

is expressed in diseased tissue. Genetic studies have been carried out on mice linking

the 5-LOX pathway to atherosclerosis, and population genetic studies involving

humans, correlates genotypes of 5-LOX, FLAP and LTA4 hydrolase to cardiovascular

disease, as shown in studies by Dwyer et al. [127] and Helgadottir et al.

[128][129][130].

The results obtained from the leaves and barks of the plant indicate good anti-

inflammatory activity via 5-LOX inhibition cascade in the chloroform extract of the

barks (IC50=29.53±0.0 ȝJPOZKHQFRPSDUHG WR1'*$ (IC50=29.19±0.02 ȝJPO

Moderate activity was shown in BE and LE while poor activity was depicted in LH

and BH in accordance with measure of activity by Kamatou et al. [94], [IC50 <30

µg/ml: good activity; 30 < IC50 <80 µg/ml : moderate activity; IC50 >80 µg/ml : poor

activity].

126

COX (prostaglandin H2 synthase) inhibition is the mechanism of action of most

NSAIDS. COX-1 is a constitutive form of the enzyme that has been linked to the

production of physiologically important prostaglandins that may play a role in

homeostasis (gastric, renal, etc). COX-2 is a form of the enzyme that is inducible by

cytokines and growth factors. Induction of COX-2 is linked to inflammatory cell

types and tissues. There is an ongoing effort to identify compounds that might inhibit

COX-2 in preference to COX-1 since such an agent may be safer and perhaps more

efficacious. In this study, COX activity is measured by utilizing peroxidase activity

and the electron donor TMPD, which turns blue upon reduction as a co-substrate.

Arachidonic acid is used as a substrate which must first be converted to

hydroperoxide thus yielding an indirect measure of COX activity. This assay has been

noted to be a high-thorough put method [112].

In this study, only the ethanol extract of leaves and barks showed inhibition against

COX-1 and COX-2 enzymes with IC50 below 100 µg/ml. Ethanol extract of leaves

had superior COX-1 inhibition (IC50 = 0.60±0.01µg/ml) compared to COX-2

inhibition (IC50 = 1.07±0.01 µg/ml) whereas the barks had superior COX-2 inhibition

(IC50 = 9.39±0.03 µg/ml) as opposed to COX-1 (IC50 = 11.41±0.03 µg/ml).

In order to determine the inhibitory selectivity on COX-1 and COX-2 enzymes, the

COX-1/COX-2 ratio was applied. According to Burnett et al. [131], A COX-1

selective inhibitor will have ratio <1, whereas a COX-2 selective inhibitor will have

ratio of > 1. In this study ethanol extract of bark was COX-2 selective (ratio 1.22)

while the leaves were COX-1 selective (ratio 0.56). The COX-2 selective behaviour

127

of the ethanol extract of the barks was similar to positive control indomethacin (ratio

1.11). This indicates the presence of lead compound that would have a more potent

COX-2 selectivity than indomethacin in the ethanol extract of barks. In view of dual

inhibition activity, the ethanol extract of barks seemed to have moderate inhibition

against 5-LOX and good inhibition against COX enzymes (selective COX-2).

Chloroform extract of the bark though had good 5-LOX inhibition but did not have

activity against COX enzymes below 100 µg/ml.

5.5 Conclusion

A combination of anti-inflammatory and antioxidant activity constitutes a good

indication on potential anti-inflammatory activity of a drug [132][133]. Keeping this

in mind the ethanol fraction of the plant warranted further studies. The pathway

mechanism of the dual inhibition of the fraction against 5-LOX and COX needs to be

investigated. However it would be interesting to investigate the bioactive component

of the chloroform extract of barks as it exhibited an activity similar to our positive

control. The clinical significance of these data is quite clear that they support a role

for Canarium patentinervium Miq. (Burseraceae Kunth.) as a source of lead

compounds in the management of inflammatory diseases.

128

CHAPTER 6

ANTI-ACETYLCHOLINESTERASE ACTIVITY

6.1 Introduction

6.1.1 Pathophysiology of $O]KHLPHU¶VGLVHDVH

Neurodegenerative disease is a term applied to a variety of conditions arising from a

chronic breakdown and deterioration of the neurons, particularly those of the central

nervous system [134] $O]KHLPHU¶V GLVHDVH $' ZDV ILUVW GHVFULEHG LQ E\ D

Bavarian neuropsychiatrist Alois Alzheimer [135]. It is a complex, multifactoral,

progressive, neurodegenerative disease primarily affecting the elderly population and

is estimated to account for 50 ± 60 % of dementia cases in persons over 65 years of

age [136]. The pathophysiology of AD is complex and involves several different

biochemical pathways.

The first neurotransmitter defect discovered in AD involved acetylcholine (ACh),

which plays an important role in memory and learning. In the CNS, ACh stimulation

of the nicotinic receptors appears to be associated with cognitive function. Normally,

ACh is stored in the nerve terminals, in structures called vesicles and is released from

the nerve endings when the nerve terminal is depolarized, thereby entering the

synapse and binding to the receptor [137]. However, in patients with AD, the ACh

which is released has a very short half-life due to the presence of large amounts of the

enzymes; acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which are

129

both present in the brain and are detected among neurofibrillary tangles and neuritic

plaques [138][139]. These enzymes hydrolyse the ester bond in the ACh molecule,

leading to loss of stimulatory activity. Approaches to enhance cholinergic function in

AD have included stimulation of cholinergic receptors or prolonging the availability

of ACh released into the neuronal synaptic cleft by use of agents which restore the

level of acetylcholine through inhibition of both AChE and BChE.

Recently, Hodges [140] demonstrated that the inhibition of AChE holds a key role not

only to enhance cholinergic transmission in the brain but also to reduce the

DJJUHJDWLRQ RI ȕ-amyloid and the formation of the neurotoxic fibrils in AD.

Therefore, AChE and BChE inhibitors have become remarkable alternatives in

treatment of AD [139]. Existing anticholinesterase drugs (example, tacrine, donepezil,

physostigmine, galantamine and heptylphysostigmine) for the treatment of dementia

are reported to have several dangerous adverse effects such as hepatotoxicity, short

duration of biological action, low bioavailability, adverse cholinergic side effects in

the periphery and a narrow therapeutic window [141][142]. This necessitates the

interest in finding better AChE inhibitors from natural resources.

The history of drug discovery has shown that plants contain active compounds that

have become new sources to investigate for the pharmaceutical industry. Plant

constituents may not only act synergistically with other constituents from the same

plant but may also enhance the activity of compounds or counteract toxic effects of

compounds from other plant species [143]. In traditional practices, numerous plants

have been used to treat cognitive disorders, including neurodegenerative diseases and

different neuropharmacological disorders [144].

130

6.1.2 Acetylcholine and its functions

Acetylcholine (ACh), a neurotransmitter in the brain plays a critical role in the

function of learning and memory. ACh is synthesized from acetyl-CoA and choline

by choline acetyltransferase, and is released into the synaptic cleft which then is

hydrolyzed by AChE to become choline and acetic acid (Figure 6.1). Choline is taken

up again into the presynaptic neurons for use in ACh synthesis. Free choline within

the nerve terminal is acetylated by a cytosolic enzyme, choline acetyltransferase

(CAT), the source of the acetyl groups being acetyl-CoA [141].

The rate-limiting process in the Ach synthesis appears to be the choline transport, the

activity of which is regulated according to the rate at which ACh is released. ACh has

functions both in the peripheral nervous system (PNS) and in the central nervous

system (CNS) as a neuromodulator [141]. Its receptors have very high binding

constants. In the peripheral nervous system, acetylcholine activates muscles, and is a

major neurotransmitter in the autonomic nervous system. In the central nervous

system, ACh and the associated neurons form a neurotransmitter system, the

cholinergic system, which tends to cause anti-excitatory actions. Cholinesterase is

present in the presynaptic nerve terminals and Ach is continually being hydrolysed

and re-synthesised [145][146].

6.1.3 Distribution and function of cholinesterase

There are two distinct types of cholinesterase both belonging to the family of serine

hydrolases, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE),

131

related closely in molecular structure but differing in their distribution, substrate

specificity and functions [145].

AChE is widely distributed in the central nervous system (CNS) and the peripheral

nervous system AChE is one of the most efficient enzymes of the nervous system

which is bound to the basement membrane in the synaptic cleft at cholinergic

synapses, where its function is to hydrolyse the released transmitter. AChE is quite

specific to acetylcholine (Ach). BChE has a widespread distribution, being found in

tissues such as liver, skin, brain and gastrointestinal smooth muscles, as well as

soluble form in the plasma. It is not particularly associated with cholinergic synapses

and has broader substrate specificity than AChE. It hydrolyses butyrylcholine more

rapidly than ACh [145][147].

6.1.4 Cholinesterase inhibitors

The most important drugs that enhance cholinergic transmission act either by

inhibiting cholinesterase or by increasing acetylcholine release by AChE. The choline

released in the process is reused in synthesizing new ACh. Inhibition of AChE increases the

amount of ACh available for neurotransmission.

There are three main effects of cholinesterase inhibition:

x effects on autonomic cholinergic synapses

x effects on neuromuscular junction

x effects on the central nervous system (CNS)

132

aptic cell

Na+ Choline

Transporter

f

AChE +

Figure 6.1: Cholinergic neurotransmission. The neurotransmitter Ach is synthesised in

presynaptic cholinergic neurons by choline acetyltransferase (CAT or ChAT). The process

entails transfer of an acetyl group from acetyl-coenzyme A to choline. Until needed, the ACh

molecules are stored in discrete vehicles at the ends of the presynaptic neurons. Arrival of a

nerve impulse triggers the release of Ca2+ ions, which activate actin microfilaments that in

turn pull the storage vesicles into position for ACh release. In a single event the vesicles

empty their contents into the synaptic cleft. Most of these molecules bind to cholinergic

receptors on adjacent postsynaptic neurons. Any that remain unbound are rapidly hydrolysed

by AChE. The choline released in the process is reused in synthesizing new ACh. Inhibition

of AChE increases the amount of ACh available for neurotransmission.

Presynaptic

cell

Choline

ACh

ACh

ChAT

ACh

Acetyl CoA

+

Choline

Acetic acid

ACh

Transporter

Postsynaptic cell

Ach receptors

133

Autonomic effects include bradycardia, hypotension, excessive secretions,

bronchoconstriction, gastrointestinal hypermotility and decrease of intraocular

pressure. This inhibition can be useful in the treatment of glaucoma that has increased

intraocular pressure. Drugs that have been used for this condition are physostigmine

and ecothiopate as eye drops. Neuromuscular action causes muscular fasciculation

and increased twitch tension and can produce depolarisation block [147] .

The clinical uses of this action would be in myasthenia gravis where transmission

fails due to too few ACh receptors due to immunological reasons, and cholinesterase

inhibition improves transmission. Drugs targeting this action would be neostigmine

and pyridostigmine taken orally. Cholinesterase inhibition at CNS has been used to

alleviate the cholinergic deficiency associated with Alzheimers disease (AD).

Although the overall AChE activity is reduced, it is increased in the neuritic plaque

and neurofibrillary tangles at the early stages of a AD patient brain [146]. It has been

VXJJHVWHG WKD\ $&K(PD\ SURPRWH DJJUHJDWLRQ RI $EHWD ȕ-amyloid) into a more

toxic amyloid form. Thus inhibiting AChE activity might increase ACh

neurotransmission in the synaptic cleft of the brain and diminish the Abeta burden

resulting in improved cognitive function and alleviating the process of amyloid

deposition. Among several existing hypotheses explaining the origin of AD, including

the cholinergic, tau and amyloid theories [148][149], the cholinergic one is most

studied. Majority of drugs on the market are thus AChE inhibitors like donepezil,

tacrine, rivstigmine and galathamine [147].

134

6.1.5 Plants as potential anticholinesterase inhibitors

In traditional practices, numerous plants have been used to treat cognitive disorders,

including neurodegenerative diseases and different neuropharmacological disorders.

Ethnopharmacological approach and bioassay-guided isolation have provided a lead

in identifying potential AChE inhibitors from plant sources, including those for

memory disorders. A variety of plants has been reported to show AChE inhibitory

activity and so may be relevant to the treatment of neurodegenerative disorders such

as AD [144].

Majority of studies have focused on the anticholinesterase alkaloids, such as physostigmine

and galantamine. So far, more than 35 alkaloids have been reported to have AChE inhibitory

activity [144]. The other major classes of compound reported to have such activity are the

terpenoids, glycosides and coumarins. Plants belonging to families Acanthaceae,

Apocynaceae, Amaryllidaceae, Angelicae, Araceae, Asclepiadaceae, Berberidaceae,

Buxaceae, Combretaceae, Compositae, Coniferae, Cyperaceae, Ebenaceae, Ericaceae,

Euphorbiaceae, Fumariaceae, Gentianaceae, Guttiferae, Lamiaceae, Leguminosae, Lilliaceae,

Lycopodiaceae, Malvaceae, Magnoliaceae, Menispermaceae, Molluginaceae, Moraceae,

Musaceae, Nelumbonaceae, Papaveraceae, Piperaceae, Rubiaceae, Rutaceae, Sapotaceae,

Solanaceae and Tamaricaceae have been reported to have AChE inhibitory potential [149].

The present study was undertaken to screen the anti-acetylcholinesterase potential of

Canarium patentinervium Miq. from family of Burseraceae. No anti-

acetylcholinesterase studies have been reported on this species to date.

135


6.2.1 Anti-acetylcholinesterase determination assays


Acetylcholinesterase (AChE) inhibitory activity was measured by slightly modifying the

spectrophotometric method developed by Ellman et al. [150]. Acethylcholine iodide which

is the substrate is hydrolysed by acethylcholinesterase enzyme resulting in the

formation thiocholine that reacts with dithiobisnitrobenzoate ion (DTNB) resulting in

yellow 5-thio-2-nitrobenzoate anion observed at a wavelength of 412 nm.

acethylcholine thiocholine + acetate

Thiocholine + dithiobisnitrobenzoate yellow color

This is accomplished by the continuous reaction of the thiol ZLWK ¶ dithiobis-2-

nitrobenzoate ion (I) to produce the yellow anion of 5-thio-2-nitro-benzoic acid (II).

H2O + (CH3)3N+CH2CH2SCOCH3 (enzyme) (CH3)3N

+CH2CH2S-

+ CH3+COO- + 2H+

(CH3)3N+CH2CH2S

- + RSSR (I) (CH3)3N+CH2CH2SSR

+ RS- (II)

R =

The reaction with the thiol has been shown to be sufficiently rapid so as not to be rate limiting

in the measurement of the enzyme, and in the concentrations used does not inhibit the

enzymic hydrolysis.

136

6.2.1.2 Protocol

5,5´-Dithio-bis(2-nitrobenzoic) acid (DTNB), galanthamine, nordihydroguairetic acid

(NDGA) and electric eel acetylcholinesterase (AChE) (Type-VI-S, EC 3.1.1.7) purchased

from Sigma AldrichR was used. Samples were dissolved in dimethyl sulfoxide (DMSO,

R&M) prior to assay and serial dilutions were done. In brief, 130 µL of 0.1 mM sodium

phosphate buffer (pH 8.0), 20 µ/ RI '71% ȝ/ RI WHVt solution and 20 µL of AChE

solution were added by multichannel automatic pipette (Eppendorf, Germany) in a 96-well

microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the

addition of 10 µL of acetylthiocholine iodide. The hydrolysis of acetylthiocholine iodide was

monitored by the formation of the yellow 5-thio-2-nitrobenzoate anion as a result of the

reaction of DTNB with thiocholines, catalyzed by enzymes at a wavelength of 412 nm

utilizing a 96-well microplate Thermo Scientific Varioskan Flash microtiter plate

reader, linked to a computer equipped with (SkanIt Software 2.4.3).

Percentage inhibition of AChE was determined by comparison of rates of reaction of

samples relative to blank sample (ethanol in phosphate buffer pH=8) using the

formula (E-S)/E x 100, where E is the activity of enzyme without test sample and S is

the activity of enzyme with test sample. The experiments were done in triplicate.

Galanthamine was purchased from Sigma (St. Louis, MO, USA) and used as

reference.

137





and SD values obtained from a minimum of three determinations. Non-linear best fit



this software. Data were analyzed using one way Anova followed by Tukey test using

GraphPad Prism5 software. A significant difference was considered at the level of P <

0.01.

6.3 Results

In the anti-acethylcholinesterase assay, chloroform extract of the barks displayed the

best activity (IC50 = 88.59±0.14 ȝJPO as opposed to galanthamine (IC50 = 0.74±0.06

ȝJPO)LJXUH7KHHWKDQROH[WUDFWRIEDUNVDQGOHDYHVIROORZWKURXJKZLWKIC50

= 186.00±0.15 ȝJPODQGIC50 = 201.24±0.15 ȝJPOUHVSHFWLYHO\. Hexane extracts of

bark and leaves and the chloroform extract of leaves had the lowest enzyme inhibition

activity (IC50 = 570.00±0.08 ȝJPO IC50 = 842.00±0.25 ȝJPO DQG IC50 =

1780.00±0.24 ȝJPOUHVSHFWLYHO\. Thus the potency of activity against AChE was BC

> BE > LE > BH >LH > LC (Table 6.1).

138

Extracts Anti-acetylcholinesterase assay, IC50ȝJPOLH 842.00±0.25

LC 1780.00±0.24

LE 201.34±0.15

BH 570.00±0.08

BC 88.59±0.14

BE 186.00±0.15

Galantamine 0.74±0.06

LH: leaf hexane extract, LC: leaf chloroform extract, LE: leaf ethanol extract, BH: bark hexane extract, BC: bark

chloroform extract, BE: bark ethanol extract. Data were obtained from three independent experiments, each

performed in triplicates (n=9) and represented as mean ± SD.Values with the same capital letter are not

significantly different (P<0.01) according to Tukey multiple comparison test.

Table 6.1. Anti-acetylcholinesterase values of Canarium patentinervium Miq.

139

0 50 100 150 200

0

20

40

60BC


% in

hibi

tion

of A

ChE

Figure 6.2: AChE inhibition by BC.

140

0 2 4 6

0

20

40

60

80

100

GL


% in

hibi

tion

of A

ChE

Figure 6.3: AChE inhibition by galanthamine.

141

6.4. Discussion

This assay measures the inhibition properties against AChE, which is the key enzyme

in the hydrolysis of acetylcholine that is responsible for muscle and organ relaxations.

Acetylcholinesterase inhibitors are used medicinally to treat myasthenia gravis to

LQFUHDVHQHXURPXVFXODU WUDQVPLVVLRQDQG WR WUHDW$O]KHLPHU¶VGLVHDVH GHILFLHQF\ LQ

the production of acetylcholine). Furthermore, oxidative and inflammatory processes

are among the pathological features associated with the central nervous system in

AlzheiPHU¶VGLVHDVH [151]. The brain of patients suffering from AD is said to be under

oxidative stress as a result of perturbed ionic calcium balances within their neurons

and mitochondria [152][153]. Accumulating evidence suggests that oxidative damage

to neurons plays an important role in the AD pathogenesis [154].

Thus, efforts to reduce oxidative injury may prove beneficial in retarding or

preventing the onset and progression of AD in patients. In this study (Chapter 4), BC

had displayed good antioxidant potential via enzymatic and non-enzymatic assays.

This in addition to its anti-acetylcholinesterase activity exhibited in this chapter

suggest that chloroform extract of the barks hold lead compounds that inhibit

acetylcholinesterase activity as well as reduce the oxidative stress with possible

neuroprotective effects. Moreover, there is evidence that AChE inhibitors have an

anti-inflammatory role through action against free radicals and amyloid toxicity, as

well as through decreasing release of cytokines from activated microglia in the brain

and blood [153].

There is an established link between the cholinergic system and inflammation as

acetylcholine, the principle neurotransmitter, is reported to attenuate the release of

142

cytokines in the parasympathetic anti-inflammatory pathway by which the brain

modulates systemic inflammatory responses to endotoxin [155]. Destruction of

neurons due to inflammation around Abeta plaques is thought to be a major factor in

the pathogenesis of AD [2]. In Chapter 5 it has been found that BC had an anti-

inflammatory action against 5-LOX (IC50 = 29.53±0.03 µg/ml) against the positive

control NDGA ((IC50 = 29.19±0.02 µg/ml). This in relation to the anti-

acetylcholinesterase activity exhibited herein suggest that BC may also acts as an anti-

inflammatory agent related to AD pathogenesis.

Because of the unclear pathogenesis of AD, there have been several hypothesis

associated with the disease such as amyloid-ȕ peptide-containing plaque formation,

excess metal ions, oxidative stress, and reduced acetylcholine levels. As proposed by

Zhang et al. [156], finding more than one approach with multifunction for AD

treatment draws attention to researchers. Thus, BC that exhibits good antioxidant

potential, anti-inflammatory and anti-acetylcholinesterase action warrants further

study for isolation of its bioactive principle.

6.5 Conclusion

The creation of effective therapeutic agents for AD would be a major medical

milestone. Different causation targets are being targeted and medicinal,

pharmacological, clinical and pathological research is on-going around the world. The

anti-acetylcholinesterase activity of Canarium patentinervium Miq. in agreement with

its potent antioxidant and anti-inflammatory activity demonstrated in Chapter 4 and 5

143

respectively represents an exciting scaffold from which to develop leads for treatment

of neurodegenerative disease.

144

CHAPTER 7

ANTIMICROBIAL ACTIVITY

7.1 Introduction

7.1.1 Infectious disease

Since their introduction, antimicrobials (antibiotics) have played an essential role in

decreasing morbidity due to infectious diseases. Antimicrobials are used for treatment

of infections and for prophylaxis against infections in humans and animals, for growth

promotion in food animal rearing and in agriculture. However infectious diseases

remain the leading cause of death worldwide and bacteria have become more resistant

to conventional antibiotic in recent years. The widespread use of these compounds is

thought to further encourage the emergence of antimicrobial resistance [157]. The

number of resistant pathogenic bacteria grows at an alarming rate worldwide and the

search for novel antimicrobial agents from medicinal plants to combat such pathogens

has become crucial for avoiding the emergence of untreatable bacterial infections

[158][159].

7.1.2 Chemotherapeutic agents: Factors affecting their effectiveness

The ideal chemotherapeutic agent has a high therapeutic index with selective toxicity,

thereby resulting in lethal damage to pathogens through the inhibition of cell wall

synthesis, protein synthesis or nucleic acid synthesis, as well as through the disruption

145

of the cell membrane and the inhibition of certain essential enzymes. This results in

selective disruption of the specific structure and/or function essential to bacterial

growth and survival, without causing similar effects to its eukaryotic host [160].

The efficacy of antimicrobial agents is influenced by a number of factors. Firstly, it is

of obvious importance that the antimicrobial agent reaches the site of the infection.

This greatly depends on the stability of the drug, its lipophilic or hydrophilic nature,

its absorption from a specific site and the presence of blood clots or necrotic tissue,

the latter of which may protect the pathogen against the antibiotic. Secondly, the

susceptibility of the pathogen to the particular chemotherapeutic agent is of utmost

importance, as well as the specific growth phase in which the pathogen is in at that

particular stage [160].

7.1.3 Drug resistance

Bacteria have evolved many different mechanisms of resistance. These can be

classified as: a) alteration in, or addition of, the target site of antimicrobial binding; b)

alteration in access to the target site for instance the decreased permeability of cell

wall or efflux mechanisms; and c) inactivation of the antimicrobial binding.

Furthermore resistance may arise through mutation or by acquisition of resistance

genes by horizontal transmission from another bacterial species [157].

On the other hand, drug resistance may be brought about by the limited drug diffusion

into the biofilm matrix, enzyme-mediated resistance, genetic adaptation, efflux

pumps, as well as through the adaptation of the outer microbial membrane, the latter

146

occurring either through the lack of or through the overexpression of certain

membrane proteins [161].

This phenomenon of increased drug resistance, combined with the multiplicity of side

effects by existing agents and the emergence of diseases for which no treatment yet

exists, makes the search for the new antimicrobial agents a highly relevant and

important subject for research. For centuries, plants have been used in the traditional

treatment of microbial infections. This assembly of knowledge by indigenous peoples

about plants and their products continue to play an essential role in health care of a

great proportion of the population [162].

7.1.4 Natural products and their role in drug discovery

Natural products have played a pivotal role in the discovery of antimicrobial drugs,

with the drug either being completely derived from the natural product, or serving as a

lead for novel drug discovery. Most antimicrobials discovered during the past 6-7

decades have been discovered through screening of soil samples, of which the

antimicrobial efficacies were determined first in vivo and later in vitro [162].

Plants synthesize a diverse array of secondary metabolites, which play a key role in

the natural defence mechanisms employed by the plant against predation by

microorganisms and insects. It is thus no surprise that these aromatic compounds

have, in numerous instances, been found to be useful antimicrobial phytochemicals

and, as a result, these compounds are now divided into different chemical categories:

147

phenolics, terpenoids and essential oils, alkaloids, lectins and polypeptides, as well as

polyacetylenes [163]. An increase in the isolation and identification of such

compounds may thus contribute greatly to the success in antibiotic discovery.

7.1.5 Canarium species and their known antimicrobial activity

Studies conducted on Canarium species have suggested that these species may well

be active against micro-organisms. Antibacterial activities was previously reported in

Canarium schweinfurthii Engl. Dichloromethane extract of Canarium schweinfurthii

Engl. had bactericidal activity against Gram-negative Vibrio cholerae with minimum

inhibitory concentration (MIC) of 0.62 mg/ml while the ethylacetate extract was

active against Gram-positive and Gram-negative bacteria namely Staphylococcus

aureus and Proteus vulgaris with MIC values of 10 mg/ml and 5 mg/ml respectively.

Ethanol extract was active against Gram-negative Vibrio cholerae and Proteus

vulgaris with MIC values of 0.62 mg/ml and 10 mg/ml respectively. The oil was also

fungicidal against Candida albicans with an inhibition zone of 23 mm [28]. In a

separate disc diffusion assay, the essential oil of Canarium schweinfurthii Engl.

abrogated the survival of Gram-negative Salmonella enterica, Gram positive

Streptococcus pyogenes and Staphylococcus aureus with an inhibition zone of 27 mm,

25 mm and 18 mm respectively [25]. Canarium patentinervium Miq. have been used

traditionally in healing wounds [164], as such this study will attempt to validate these

ethnopharmacological claims.

148

7.2 Materials and Methods

The in vitro antimicrobial properties of Canarium patentinervium Miq. was evaluated

using the disc diffusion assay, minimum inhibitory concentration (MIC) assay,

minimum bactericidal assay (MBC) and death kinetic assay. The hexane, chloroform

and ethanol extracts of Canarium patentinervium Miq. were prepared (see Chapter 2

for the extraction process). The following bacterial strains were employed in the

screening:

i. Gram-positive bacteria such as Staphylococcus aureus (ATCC 11632),

Bacillus cereus (ATCC 10876), methicillin-resistant Staphylococcus

aureus (ATCC 43300) and Gram-negative bacteria such as Escherichia

coli (ATCC 10536) and Pseudomonas aeruginosa (ATCC 10145).

ii. Ten clinical bacterial strains and 3 clinical strain of yeasts obtained from

the Bacteriology Unit, Department of Microbiology and Immunology of

UKM Medical Centre. The assays involving these clinical strains were

also perfomed at HUKM.

7.2.1 Disc diffusion assay

7.2.1.1 Principle of method

The principles of determining the effectivity of a noxious agent to a bacterium were

well enumerated by Rideal, Walker and others at the turn of the century. The

discovery of antibiotics made these tests (or their modification) too cumbersome to be

put up as a routine due to the large number of tests it requires [165]. The ditch plate

149

method of agar diffusion used by Alexander Fleming was the forerunner of a variety

of agar diffusion methods devised by workers in this field. The Oxford group used

these methods initially to assay the antibiotic contained in blood by allowing the

antibiotics to diffuse out of reservoirs in the medium in containers placed on the

surface [165]. With the introduction of a variety of antimicrobials it became necessary

to perform the antimicrobial susceptibility test as a routine.

In this assay, the antimicrobial contained in a reservoir was allowed to diffuse out into

the medium and interact in a plate freshly seeded with the test organisms. Even now a

variety of antimicrobial containing reservoirs are used but the antimicrobial

impregnated absorbent paper disc is by far the commonest type used. The disc

diffusion method of antimicrobial susceptibility testing (AST) is the most practical

method and is still the method of choice for the average laboratory. Automation may

force the method out of the diagnostic laboratory but in this country as well as in the

smaller laboratories of many countries, it will certainly be the most commonly carried

out microbiological test for many years to come [165].

The Kirby-Bauer and Stokes' methods are usually used for antimicrobial susceptibility

testing, with the Kirby-Bauer method being recommended by the National Committee

for Clinical Laboratory Standards (NCCLS). The accuracy and reproducibility of this

test are dependent on maintaining a standard set of procedures. NCCLS is an

international, interdisciplinary, non-profit, non-governmental organization composed

of medical professionals, government, industry, healthcare providers, educators etc. It

promotes accurate antimicrobial susceptibility testing (AST) and appropriate reporting

by developing standard reference methods, interpretative criteria for the results of

150

standard AST methods, establishing quality control parameters for standard test

methods, provides testing and reporting strategies that are clinically relevant and cost-

effective [165]. Interpretative criteria of NCCLS are developed based on international

FROODERUDWLYHVWXGLHVDQGZHOOFRUUHODWHGZLWK0,&¶VDQGWKHUHVXOWVKDYHFRUURERUDWHG

with clinical data. Based on study results NCCLS interpretative criteria are revised

frequently. NCCLS is approved by FDA-USA and recommended by WHO

[165][166].

7.2.1.2 Protocol [166][167][168]

i. Preparation of Mueller Hilton Agar

Mueller Hilton Agar was prepared from a commercially available dehydrated base

DFFRUGLQJWRWKHPDQXIDFWXUHU¶VLQVWUXFWLRQV The agar solution was then autoclaved.

It was then allowed to cool in a 45-50 oC water bath. The agar was then poured into

plastic flat bottomed petri dishes on a level, horizontal surface to give a uniform depth

of approximately 4 mm. This corresponds to 25 ml for plates with a diameter of 100

mm. The agar medium was then allowed to cool to room temperature and unless plate

is used the same day, stored in a refrigerator (2 to 8 oC). A representative sample of

each batch of plates was examined for sterility by incubation at 30-35 oC for 24 hrs or

longer.

ii. Preparation of dried filter paper discs/ cotton swabs/ tryptic soy broth/

normal saline solution

Whatman filter paper no.1 was used to prepare discs approximately 6 mm in diameter.

They are then placed in a bijou bottle and autoclaved for sterility. Same was done for

151

cotton swabs. Tryptic soy broth (TSB) (20 ml) ZDVSUHSDUHG DV WKHPDQXIDFWXUHU¶V

instructions. About 5 ml was poured into a Bijou bottle and autoclaved. About 150

ml of sodium chloride solution of 0.9 % was prepared and autoclaved

iii. Preparation of sample extracts, negative control and standard antibiotic

solution.

Sample extracts were dissolved in DMSO at a concentration of 100 mg/ml and

filtered. Negative control was DMSO. Standards used were ampicillin and

streptomycin which were prepared at the concentration of 100 µg/ml.

iv. Preparation of fresh/ pure colonies of bacteria

Tryptic soy agar was prepared according to the manufacturers intructions. Agar was

autoclaved and poured on petri dishes to 4 mm, approximately 25 ml to a 100 mm

petri dish. Bacterias are streaked on the tryptic soya agar (TSA) plates in 4 density

level around the plates. Plates are then sealed with a parafilm and incubated for 18 hrs

to obtain single colonies.

v. Preparation of disc with extracts, control and standards

About 10 µl of sample extracts was pipetted onto the disc in triplicates. Same was

done for controls and standards. Impregnated discs are left to air dry overnight for 12

hrs.

vi. Inoculum Preparation

At least 3-5 well isolated colonies of the same morphological type are selected from

an agar plate culture. The top of each colony was touched with a sterilized loop and

152

the growth was transferred into the Bijou bottle containing TSB. This was repeated

for each bacteria into each Bijou bottle. The broth culture was incubated at 35 oC for

about 2 hrs till it achieved the turbidity of 0.5 Mcfarland standard/625 nm to yield 1 X

108 cfu/ml. Using optical density at 625 nm, the reading below must be obtained for

different bacteria, turbidity was adjusted with sterile normal saline.

vii. Inoculation of test plates

The bacterial broth was used within 15 mins after the turbidity of the inoculum

suspension was adjusted. Sterile cotton swab was dipped into the suspension and

rotated several times and pressed firmly on the inside wall of the tube above the fluid

level. This was to remove any excess inoculum from the swab. The dried surface of a

Mueller Hilton agar plate was inoculated by streaking the swab over the entire agar at

every 60 oC. As a final step, the rim of the agar was swabbed.

viii. Application of discs to inoculated agar plates

Each disc was pressed down on the agar to ensure complete contact according to

prepared template. Plates were placed in 35 oC incubator within 15 mins after discs

are applied.

viiii. Reading plates and interpretation of results

After 18 hrs of incubation, each plate was examined. Zone of inhibition was measure

using sliding calipers to the nearest millimeters. The inhibition zones would thus

include disc size of 6 mm. The experiment was done three times and the mean values

are presented.

153

7.2.2 Minimum inhibitory concentration assay


The MIC is the lowest concentration being beyond the concentration where no growth

inhibition of test organisms was observed. Minimum inhibitory concentration (MIC)

assays investigate the in vitro susceptibility of organisms to antimicrobial agents. This

method employs different dilutions of the antimicrobial agent and quantitatively

investigates the lowest concentration at which visible microbial growth inhibition is

achieved. Microbial growth is visualised through the addition of the tetrazolium salts,

specifically p-iodonitrotetrazolium (INT, Sigma-Aldrich). The assay is based on the

detection of dehydrogenase activity in living cells by being converted from a

colourless solution to an intensely coloured formazan (red) product [169]. This

method yields reproducible results within one doubling dilution of the end point of the

activity.

7.2.2.2 Protocol

The MIC of the plant extracts was determined by serial dilution, as described by Eloff

[169]. These dilution experiments were performed in sterile 96-well microtiter plates.

i. Preparation of sample

Stock solutions of the respective plant extracts were prepared in 1.5 ml

microcentrifuge tubes (Eppendorff) by dissolving dry plant extract in

154

dimethylsulphoxide (DMSO, Saarchem) to a final concentration of 64 mg/ml.

$OLTXRWVRIȝORIWKHVWRFNVRlution were transferred aseptically into the top row

RIPLFURWLWHUSODWHURZ$ZKLFKDOUHDG\FRQWDLQHGȝODOLTXRWVRIVWHULOHZDWHU

thereby resulting in a 50 % dilution of the stock solution to 32 mg/ml. After adequate

mixing of the contents of eacKZHOOȝODOLTXRWVRIURZ$ZHUHWUDQVIHUUHGIURP

URZ$WRWKHFRUUHVSRQGLQJZHOOVLQURZ%DOVRFRQWDLQLQJȝODOLTXRWVRIVWHULOH

water), followed by mixing and resulting in yet another 50% dilution of the plant

extract (to 16 mg/ml). This process was repeated IRU HYHU\ URZ UHVXOWLQJ LQȝO

aliquots ranging in concentration from 32 mg/ml (row A) to 0.25 mg/ml (row H).

ii. Preparation of inocolums

In order to determine the range of antimicrobial activity of Canarium patentinervium

Miq., four different microbial (reference) cultures and 10 clinical strains were used

and are listed below.

i. Gram-positive bacteria

x Staphylococcus aureus (ATCC 11632)

x Bacillus cereus (ATCC 10876)

x Methicillin-resistant Staphylococcus aureus (ATCC 43300)

x Staphylococcus aureus MSSA

x Staphylococcus aureus MRSA

x Coagulase negative Staph: Oxacillin (S)

x Coagulase negative Staph: Oxacillin (R)

x Enterococcus faecalis

155

ii. Gram-negative bacteria

x Pseudomonas aeruginosa (ATCC 10145)

x Escherichia coli

x Klebsiella sp

x Escherichia coli ESBL

x Klebsiella pneumoniae ESBL

iii. Yeast

x Candida parapsilosis

Bacterias are streaked on TSA plates in 4 density level around the plate. Plates are

then sealed with a parafilm and incubated for 18 hrs to obtain fresh single colonies. At

least 3-5 well isolated colonies of the same morphological type are selected from the

agar plate culture. The top of each colony was touched with a sterilized loop and the

growth was transferred into Bijou bottle containing TSB/MHB. This was repeated for

each bacteria placed into each Bijou bottle. Broth was incubated at 35 oC for about 2

hrs till it achieved 0.5 Mcfarland Standard/ 600 nm (OD-0.1 nm) to yield 1 x 108

cfu/ml [170]. Turbidity was adjusted with sterile normal saline. To obtain 10 x 105

cfu/ml, add 100 µl of adjusted broth to 9900 µl of TSB/MHB (1:100 dilution).

iii. MIC assay

7KLVZDVIROORZHGE\DGGLWLRQRIȝODWconcentration of 10 x 105 colony forming

units (cfu)/ml, of liquid microbial culture grown in Mueller Hilton Broth (MHB) to

HDFK ZHOO 7KLV \LHOGHG D ILQDO YROXPH RI ȝO LQ HDFK ZHOO DQG ILQDO H[WUDFW

156

concentrations ranging from 16 mg/ml in row A to 0.125 mg/ml in row H (Figure 4.1)

with bacteria 5 x 105 cfu/ml. The microtiter plates were incubated at 37 °C, overnight

for bacteria and 48 hours for yeasts (as yeasts require a greater time period for

growth). After incubation at 37 &ȝORIDPJPOVROXWLRQRI,17ZDVDGGHGWR

each well as an indicator of microbial growth. The plates were incubated at 37 oC for

30 min (bacteria) and 24 hr (yeast) and the MIC values visually determined. The

lowest concentration of each extract displaying no visible growth was recorded as the

minimum inhibitory concentration. The concentration that inhibited bacterial/yeast

growth completely (the first clear well) was taken as the MIC value. MIC values were

determined at least in duplicate and repeated to confirm activity

In order to determine the sensitivity of the microorganisms, positive control

experiments were conducted: (1) for bacterial strains, ampicillin (Sigma-Aldrich) at a

starting concentration of 0.10 mg/ml in sterile water, and (2) for yeast strains,

Amphotericin B (Sigma-Aldrich), at a starting concentration of 0.10 mg/ml in DMSO

and water (where 1.00 mg/ml was prepared in DMSO, and diluted to 0.10 mg/ml in

sterile water thereafter). The final concentrations for these experiments ranged from

25.00 µg/ml (row A) to 0.19 µg/ml (row H). A negative control experiment was

conducted using only DMSO.

157

7.2.3 Minimum bactericidal concentration (MBC)


Minimum bactericidal concentration (MBC) was recorded as a lowest extract

concentration killing 99.9 % of the bacterial inocula after 24 hr incubation at 37 °C.

Each experiment was repeated at least three times.

7.2.3.2 Protocol

The determination of MBC was performed using the method of Ozturk & Ercisli

[171] only for the susceptible bacterias/ fungi from the MIC assay (Section 7.2.2).

Ten microliters were taken from the well obtained from the MIC experiment (MIC

value) and two wells above the MIC value well and spread on MHA plates. The

number of colony was counted after 18-24 hrs of incubation at 37 °C. The

concentration of sample that produces < 10 colonies was considered as MBC value.

7.2.4 Time-kill assay


Death kinetic studies are often referred to as time-kill studies, and are used to

determine the rate at which the antimicrobial agent kills pathogens over time, as well

as the extent at which the activity occurs. Upon introduction of a micro-organism into

a new environment, its growth displays a lag phase during which no cell growth

158

occurs, after which its growth enters the exponential phase, where microbial cell

multiplication occurs at an exponential rate (doubling in number at regular intervals).

This exponential phase is followed by the stationary phase, the latter of which usually

results from the depletion of nutrients (carbon, nitrogen and/or oxygen sources) [160].

However, upon exposure to a constant concentration of an antimicrobial agent, the

organism will remain within the lag phase for a certain amount of time. This is

followed by a log-linear killing phase, during which the number of microbial colonies

are decreased until it enters into a second lag phase. Re-growth may occur after this

second lag phase, but documentation of this phenomenon rarely occurs as time-kill

studies are usually performed over a 24 hour period only [172][173].

7.2.4.2 Protocol

Only samples that have MIC values of lower or equivalent to 0.5 mg/ml (strong

inhibitors) are tested for the time-kill [174]. Concentration of samples to be tested is

determined base on the MIC values (up to 4 times the MIC value) [175]. Samples and

incolums are prepared as in section 7.2.2.2 (i-ii). This was followed by addition of

ȝORIPLFURELDOEURWKDWconcentration of 10 x 105 colony forming units (cfu)/ml,

of liquid microbial culture grown in Mueller Hilton Broth (MHB) to each well. This

\LHOGHG D ILQDO YROXPH RI ȝO LQ HDFK ZHOO of the extract. The plates are then

incubated at 37 °C and optical density was recorded at 1 hr intervals up to 18 hrs at

wavelength of 600 nm. Graphs were plotted on the basis of the turbidity varying over

a period of time. The growth rate thus obtained was studied for any signs of

bactericidal effects of the plant extract. Ampicillin was used as positive control. A

159

solution of the solvent in which dried extract was dissolved served as negative

control.


Growth curve of bacteria were analysed using MS-Excel and data were reported as

mean and SD values obtained from a minimum of three determinations. Bacterial

growth curve was plotted with SD and 95% confidence interval. All data were

expressed as mean ± standard deviation. Data were analyzed using one way Anova

followed by Tukey test. A significant difference was considered at the level of P <

0.01.

7.3 Results


The six extracts of Canarium patentinervium Miq. that were screened for

antimicrobial activity using the disc diffusion assay displayed good activity (Table

7.1). The extracts were screened for activity against Gram-positive bacteria

(Staphylococcus aureus ATCC 11632, MRSA ATCC 43300, Bacillus cereus ATCC

10876, clinical strains consisting of MSSA, MRSA, Coagulase-negative

Staphyloccocus: oxacillin sensitive/resistant, Enterococcus faecalis and Streptococcus

pneumoniae), Gram-negative bacteria (Escherichia coli ATCC 10536, Pseudomonas

aeruginosa ATCC 10145, clinical strains consisting of Esherichia coli, Klebsiella sp,

160

Esherichia coli ESBL and Klebsiella pneumoniae ESBL and yeasts (Candida

albicans, Candida glabrata and Candida parapsilosis). All the bacterias and yeast

were sensitive to the extracts except Streptococcus pneumoniae, Eschericia coli

ATCC 10536, Candida glabrata and Candida albicans. The ethanol extract of leaves

and the hexane extract of bark displayed good antibacterial activity against both

Gram-positive bacteria and Gram-negative bacteria. The ethanol extracts and hexane

extract of the bark had the best sensitivity to Candida parapsilopsis (inhibition zone

14 mm).

Both ATCC and clinical strains of MSSA and MRSA were sensitive to the ethanol

extracts (9-12 mm) (Figure 7.1) and hexane extract of barks (8-14 mm). Coagulase-

negative Staphylococcus aureus: oxacillin sensitive and resistant and Enterococcus

faecalis were most sensitive to the ethanol extracts (inhibition zone 8-12 mm). Gram-

negative bacteria Klebsiella pneumoniae ESBL (inhibition zone 9-14 mm) had

sensitivity to all the bark extracts while Klebsiella sp showed sensitivity only towards

the ethanol extracts (inhibition zone 11-13 mm). Pseudomonas aeruginosa ATCC

10145 was inhibited by the ethanol extract of leaves (inhibition zone 11 mm) (Figure

7.2) and hexane extract of barks (inhibition zone 12 mm) while poor activity was

seen with Esherichia coli with all extracts.

7.3.2 MIC assay

All extracts displayed some activity against Gram-positive and Gram-negative

bacteria and yeast (Table 7.2). The activity against MSSA reference and clinical

strains ranged from 0.50 mg/ml to 2.00 mg/ml with the strongest activity seen with

161

the ethanol extract of leaves. The activity against MRSA reference and clinical strains

ranged from 0.25 mg/ml to 2.00 mg/ml with the lowest MIC seen in the ethanol

extract of leaves. All the extracts had activity against coagulase-negative

Staphylococcus aureus: oxacillin resistant and sensitive with MIC ranging from as

low as 0.50 mg/ml (ethanol extract of leaves) to 16.00 mg/ml (hexane extract of

leaves). Enterococcus faecalis had activity only against the ethanol extracts with an

MIC of 0.50 mg/ml. Bacillus cereus ATCC 10876 showed sensitivity only against the

ethanol extracts (MIC 0.5 mg/ml) and hexane extract of barks (MIC 1.00 mg/ml).

All the bark extracts showed activity against Gram-negative Klebsiella sp. with an

MIC range of 0.50 mg/ml to 4.00 mg/ml while only the ethanol extracts of leaves and

barks showed activity against Klebsiella pneumoniae ESBL with MIC of 0.5 mg/ml.

Pseudomonas aeruginosa ATCC 10145 was sensitive towards ethanol extract of

leaves and hexane extract of barks with an MIC of 1.00 mg/ml and 4.00 mg/ml

respectively. Esherichia coli clinical strain was sensitive only towards ethanol extract

of barks with an MIC of 0.50 mg/ml while Escherichia coli ESBL was inhibited by

the chloroform extracts and hexane extract of barks with MIC of 8.00 mg/ml. The

ethanol extracts and hexane extract of bark both had activity against yeast Candida

parapsilopsis with the MIC of 2.00 mg/ml and 8.00 mg/ml respectively.

7.3.3 MBC assay and MBC/MIC ratio

The action of an antibacterial on the bacterial strains can be characterized with two

parameters such as minimum inhibitory concentration (MIC) and minimum

bactericidal concentration (MBC). According to the ratio MBC/MIC, we appreciated

162

antibacterial activity. If the ratio 0%&0,& , the effect was considered as

bactericidal but if the ratio MBC/MIC > 4 , the effect was defined as bacteriostatic

[176][177]. The MBC and MBC/MIC ratio are displayed in Table 7.3 and 7.4

respectively.

Both the ethanol extract of leaf and bark were bactericidal against Bacillus cereus

ATCC 10876 (MBC of 1.00 mg/ml), MSSA ATCC 11632 (MBC range of 1.00 to

2.00 mg/ml), Klebsiella sp and Klebsiella pneumonia ESBL (MBC of 2.00 mg/ml)

and yeast Candida parapsilopsis (MBC 8.00 mg/ml). The hexane extract of barks was

bactericidal against MSSA, MRSA (both strains) and Pseudomonas aeruginosa

ATCC 10145 with MBC range of 2.00 mg/ml to 8.00 mg/ml. Chloroform extract of

barks showed bactericidal activity against MSSA (ATCC 11632 and clinical strains)

with MBC of 1.00 mg/ml and 4.00 mg/ml respectively. Both hexane and chloroform

extract of barks were bactericidal against Klebsiella sp with MBC/MIC ratio of 4.

Rest of the MBC as displayed in Table 7.3 were all having bacteriostatic action with

an MBC/MIC ratio > 4.


Only samples that have MIC values of lower or equivalent to 0.5 mg/ml (strong

inhibitors) are tested for the time-kill [174]. The results obtained for the time-kill

study of Canarium patentinervium Miq. are shown in Figure 7.3-7.22. The MBC

values (Table 7.3) was consistent with the cidal concentration as displayed in the

growth curve of this time-kill study. Blank control (broth alone) was the curve marked

as 0 mg/ml of extract. The antibacterial activity was observed at different times upon

163

exposure of bacterias to the different concentration of extracts. This observation was

made by varying turbidity over time for a period of 18 hrs.

7.4 Discussion


According to Gislene et al. [178], chemicals that have antibacterial activity with zones

of inhibition of 7 mm and above can be considered as potential antimicrobial

candidates. In the present study, antibacterial screening of six extracts of Canarium

patentinervium Miq. showed varying degrees of antibacterial activity against human

pathogenic bacteria such as Staphyloccocus aureus, MRSA, Bacillus cereus (ATCC

strain respectively), coagulase-negative Staphylococcus aureus, Enterococcus

faecalis, Klebsiella pneumoniae ESBL and yeast Candida parapsilopsis (clinical

strain respectively) cultured in vitro. Out of six extracts, the ethanol extract of leaves

and hexane extract of barks exhibited significant antibacterial activity against both

Gram-positive and Gram-negative bacteria. The best sensitivity to the ethanol extract

of the leaves at 1 mg/disc was obtained against Staphyloccocus aureus (12 mm),

MRSA (10 mm), Bacillus cereus (12 mm) and Enterococcus faecalis (12 mm),

Klebsiella pneumoniae ESBL (13 mm) and yeast Candida parasilopsis (14 mm). The

hexane extract of the barks showed best activity against MSSA (14 mm), Klebsiella

sp (14 mm) and yeast Candida parapsilopsis (14 mm).

164

This is of special interest since most Gram-negative bacteria are more resistant to

plant extracts [179]. Results from our phytochemical analysis revealed that the

ethanol extract of leaves and barks of Canarium patentinervium Miq. accumulate

substantial amounts of flavonoids and tannins which could be well correlated with the

activities measured.

165

Figure 7.1: Zone of inhibition against Staphylococcus aureus (left plate) and methicillin resistant Staphylococcus aureus (right plate) for leaf ethanol extract

at 1 mg/disc.

166

Figure 7.2: Zone of inhibition against Bacillus cereus (left plate) and Pseudomonas aeruginosa (right plate) for leaf ethanol extract at 1 mg/disc.

167

Table 7.1: Antibacterial activity of Canarium patentinervium Miq. (1 mg/disc ) vs. ampicillin against 5 ATCC bacterias and 10 clinical bacterial strains and 3 clinical yeast strains tested by disc diffusion assay

Bacteria/ yeast Plant extract (Zone of inhibition) (mm) a

LH LC LE BH BC BE Ampicilinc Amphotericinc

MSSA Staphylococcus aureus ATCC 11632 - - 12.00±0.21 11.00±0.25 7.00±0.29 9.00±0.58 10.00±0.66 NA

MRSA Staphylococcus aureus ATCC 43300 - - 10.00±0.42 9.00±0.25A - 9.00±0.00A - NA

Bacillus cereus ATCC 10876 - - 12.00±0.25B 12.00±0.45B - 9.00±0.00 8.00±0.00 NA

Eschericia coli ATCC 10536 - - - - - - 7.00±0.00 NA

Pseudomonas aeruginosa ATCC 10145 - - 11.00±0.34 12.00±0.47 - - - NA

MSSA Staphylococcus aureus - - 11.00±0.23 14.00±0.43 13.00±0.17 10.00±0.45 7.00±0.26 NA

MRSA Staphylococcus aureus - - 10.00±0.37C 8.00±0.29 - 10.00±0.47C - NA

Eschericia coli - - - - - 8.00±0.57 - NA

Eschericia coli ESBL - 7.00±0.18 - 8.00±0.38D 8.00±0.26D - - NA

Coagulase-negative Staphylococcus aureus: O (R) - 7.00±0.59 10.00±0.00 8.00±0.34E 8.00±0.36E 9.00±0.34 - NA

Coagulase-negative Staphylococcus aureus: O (S) 7.00±0.37F 7.00±0.16F 12.00±0.29G - - 12.00±0.23G 10.00±0.27 NA

Enterococcus faecalis - - 12.00±0.46 - - 8.00±0.15H 8.00±0.28H NA

Streptococcus pneumoniae - - - - - - - NA

Klebsiella sp - - 11.00±0.38I 14.00±0.00 11.00±0.34I 9.00±0.42 10.00±0.14 NA

Klebsiella pneumoniae ESBL - - 13.00±0.25 - - 11.00±0.27 - NA

Candida parapsilosis - - 14.00±0.12K 14.00±0.24K - 14.00±0.10K NA 11.00±0.23

Candida glabrata - - - - - - NA 9.00±0.15

Candida albicans - - - - - - NA -

LH-leaf hexane extract, LC- leaf chloroform extract, LE-leaf ethanol extract, BH- bark hexane extract, BC- bark chloroform extract, BE- bark ethanol extract, NA-not applicable

a: inhibition zones are the mean including disc (6 mm) b: sample extract at the concentration of 100 mg/ml (1mg/disc), c: ampicillin/amphotericin at 100 µg/ml = 1 µg/disc, -: no activity noted

Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD.

Values with the same capital letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

168

Table 7.2: MIC values for Canarium patentinervium Miq. extracts against 4 ATCC bacterias and 9 clinical bacterial strains and 1 clinical yeast strain

Bacteria/ yeast Plant extract minimum inhibitory concentration MIC (mg/ml) and positive controla MIC (µg/ml)

LH LC LE BH BC BE Ampicilina Amphotericina

MSSA Staphylococcus aureus ATCC 11632 - - 0.50±0.10 2.00±0.10 1.00±0.00A 1.00±0.00A 1.56±0.00 NA

MRSA Staphylococcus aureus ATCC 43300 - - 0.50±0.00 1.00±0.00B - 1.00±0.00B - NA

Bacillus cereus ATCC 10876 - - 0.50±0.15C 1.00±0.00 - 0.50±0.00C 6.25±0.00 NA

Pseudomonas aeruginosa ATCC 10145# - - 1.00±0.00 4.00±0.05 - - - NA

MSSA Staphylococcus aureus - - 0.50±0.10 2.00±0.00D 2.00±0.00D 0.50±0.00 >25.00±0.00 NA

MRSA Staphylococcus aureus - - 0.25±0.00E 2.00±0.10 - 0.25±0.00E - NA

Eschericia coli - - - - - 0.50±0.00 - NA

Eschericia coli ESBL - 8.00±0.00F - 8.00±0.00F 8.00±0.00F - - NA

Coagulase-negative Staphylococcus aureus: O (R) - 8.00±0.00 1.00±0.00 0.50±0.00 2.00±0.00 0.50±0.00 - NA

Coagulase-negative Staphylococcus aureus: O (S) 16.00±0.00 8.00±0.00 0.50±0.00G - - 0.50±0.10G 8.00±0.00 NA

Enterococcus faecalis - - 0.50±0.00I - - 0.50±0.00I 3.13±0.00 NA

Klebsiella sp - - 0.50±0.00J 4.00±0.00K 4.00±0.00K 0.50±0.00J >25.00±0.00 NA

Klebsiella pneumoniae ESBL# - - 0.50±0.00 - - 0.50±0.00 - NA

Candida parasilopsis - - 2.00±0.00K 8.00±0.00 - 2.00±0.00K NA 0.78±0.00

LH-leaf hexane extract, LC- leaf chloroform extract, LE-leaf ethanol extract, BH- bark hexane extract, BC- bark chloroform extract, BE- bark ethanol extract

Data were obtained from three independent experiments, each performed in duplicates (n=6) and represented as mean ± SD, -: no activity, NA-not applicable

Values with the same capital letter are not significantly different (P<0.01) according to Tukey multiple comparison test. #: statistical analysis not performed due to insufficient data

169

Table 7.3: MBC values for Canarium patentinervium Miq. extracts against 4 ATCC bacterias and 9 clinical bacterial strains and 1 clinical yeast strain

Bacteria/ yeast Plant extract minimum inhibitory concentration,MBC (mg/ml) and positive controla MBC (µg/ml)

LH LC LE BH BC BE Ampicilina Amphotericina

MSSA Staphylococcus aureus ATCC 11632 - - 2.00±0.00A 2.00±0.00A 1.00±0.00B 1.00±0.00B 6.25±0.00 NA

MRSA Staphylococcus aureus ATCC 43300 - - 2.00±0.00 4.00±0.00C - 4.00±0.00C - NA

Bacillus cereus ATCC 10876 - - 1.00±0.10D 8.00±0.00 - 1.00±0.00D >25.00±0.00 NA

Pseudomonas aeruginosa ATCC 10145 - - 4.00±0.00 8.00±0.05 - - - NA

MSSA Staphylococcus aureus - - 4.00±0.10E 4.00±0.00E 4.00±0.00E 2.00±0.00 >25.00±0.00 NA

MRSA Staphylococcus aureus - - 2.00±0.00 8.00±0.10 - 1.00±0.00 - NA

Eschericia coli# - - - - - 2.00±0.00 - NA

Eschericia coli ESBL# >16.00±0.00 - >16.00±0.00 >16.00±0.00 - - NA

Coagulase-negative Staphylococcus aureus: O (R) >16.00±0.00 4.00±0.00F 2.00±0.00 8.00±0.00 4.00±0.00F - NA

Coagulase-negative Staphylococcus aureus: O (S)# >16.00±0.00 >16.00±0.00 2.00±0.00 - - 2.00±0.10 12.50±0.00 NA

Enterococcus faecalis# - - 4.00±0.00 - - 4.00±0.00 6.25±0.00 NA

Klebsiella sp - - 2.00±0.00I 16.00±0.00J 16.00±0.00J 2.00±0.00I >25.00±0.00 NA

Klebsiella pneumoniae ESBL# - - 2.00±0.00 - - 2.00±0.00 - NA

Candida parapsilopsis# - - 8.00±0.00 >16.00±0.00 - 8.00±0.00 NA 3.13±0.00

LH-leaf hexane extract, LC- leaf chloroform extract, LE-leaf ethanol extract, BH- bark hexane extract, BC- bark chloroform extract, BE- bark ethanol extract

Data were obtained from three independent experiments, each performed in duplicates (n=6) and represented as mean ± SD, -: no activity, NA-not applicable

Values with the same capital letter are not significantly different (P<0.01) according to Tukey multiple comparison test. #: statistical analysis not performed due to insufficient data

170

Table 7.4: Bacteriostatic (-) and Bactericidal (+) effects of Canarium patentinervium Miq. extracts

Bacteria/ yeast MBC/MIC ratio (+/-)

LE BH BC BE

MSSA Staphylococcus aureus ATCC 11632 4 (+) 1 (+) 1 (+) 1 (+)

MRSA Staphylococcus aureus ATCC 43300 4 (+) 4 (+) - 4 (+)

Bacillus cereus ATCC 10876 2 (+) 8 (-) - 2 (+)

Pseudomonas aeruginosa ATCC 10145 4 (+) 2 (+) - -

MSSA Staphylococcus aureus 8 (-) 2 (+) 2 (+) 4 (+)

MRSA Staphylococcus aureus 8 (-) 4 (+) - 4 (+)

Eschericia coli - - - 4 (+)

Coagulase-negative Staphylococcus aureus: O (R) 4 (+) 4 (+) 4 (+) 8 (-)

Coagulase-negative Staphylococcus aureus: O (S) 4 (+) - - 4 (+)

Enterococcus faecalis 8 (-) - - 8 (-)

Klebsiella sp 4 (+) 4 (+) 4 (+) 4 (+)

Klebsiella pneumoniae ESBL 4 (+) - - 4 (+)

Candida parapsilopsis 4 (+) ND - 4 (+)

LE-leaf ethanol extract, BH- bark hexane extract, BC- bark chloroform extract, BE- bark ethanol extract

-: no activity, ND: not determined

171

Figure 7.3: Time-kill plot for MSSA ATCC 11632 in presence of LE

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)

0 mg/ml 1mg/ml 0.25mg/ml 0.5mg/ml 2mg/ml

172

Figure 7.4: Time-kill plot for MRSA ATCC 43300 in presence of LE

-0.2-0.1

00.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

Time (hr)

0 mg/ml 0.25mg/ml 0.5mg/ml 1mg/ml 2mg/ml

173

Figure 7.5: Time-kill plot for MSSA (clinical strain) in presence of LE

-0.10

0.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)

0 mg/ml 0.25mg/ml 0.5mg/ml 1mg/ml 2mg/ml 4mg/ml

174

Figure 7.6: Time-kill plot for MRSA (clinical strain) in presence of LE

-0.20.00.20.40.60.81.01.21.41.6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


175

Figure 7.7: Time-kill plot for MSSA (clinical strain) in presence of BE

-0.10

0.10.20.30.40.50.60.70.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


176

Figure 7.8: Time-kill plot for MRSA (clinical strain) in presence of BE

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


177

Figure 7.9: Time-kill plot for Bacillus cereus ATCC 10876 in presence of LE

-0.10.00.10.10.20.20.30.30.40.40.50.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


178

Figure 7.10: Time-kill plot for Bacillus cereus ATCC 10876 in presence of BE

-0.1

-0.1

0.0

0.1

0.1

0.2

0.2

0.3

0.3

0.4

0.4

0.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


179

Figure 7.11: Time-kill plot for for Escherichia coli (clinical strain) in presence of BE

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


180

Figure 7.12: Time-kill plot for coagulase-negative Staphylococcus aureus: O (R) (clinical strain) in presence of LE

-0.10

0.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


181

Figure 7.13: Time-kill plot for coagulase-negative Staphylococcus aureus: O (R) (clinical strain) in presence of BH

-0.10

0.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


182

Figure 7.14: Time-kill plot for coagulase-negative Staphylococcus aureus: O (R) (clinical strain) in presence of BE

-0.10

0.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in

OD

)

Time (hr)


183

Figure 7.15: Time-kill plot for coagulase-negative Staphylococcus.aureus: O (S) (clinical strain) in presence of LE

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


184

Figure 7.16: Time-kill plot for coagulase-negative Staphylococcus aureus: O (S) (clinical strain) in presence of BE

-0.10

0.10.20.30.40.50.60.70.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


185

Figure 7.17: Time-kill plot for Enterococcus faecalis (clinical strain) in presence of LE

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


186

Figure 7.18: Time-kill plot for Enterococcus faecalis (clinical strain) in presence of BE

-0.10

0.10.20.30.40.50.60.70.8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


187

Figure 7.19: Time-kill plot for Klebsiella sp. (clinical strain) in presence of LE

0.00.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


188

Figure 7.20: Time-kill plot for Klebsiella sp. (clinical strain) in presence of BE

0.00.10.20.30.40.50.60.70.80.9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


189

Figure 7.21: Time-kill plot for Klebsiella pneumonia ESBL (clinical strain) in presence of LE

0.0

0.1

0.2

0.3

0.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


190

Figure 7.22: Time-kill plot for Klebsiella pneumonia ESBL (clinical strain) in presence of BE

0.0

0.1

0.2

0.3

0.4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)


191

7.4.2 MIC, MBC and MIC/MBC ratio

Upon identifying possible antimicrobial action with the disc diffusion assay, a more

comprehensive investigation was done to evaluate the total bactericidal/ bacteriostatic

activity of an extract against a microb using the MIC/ MBC and time-kill assay. All

plant species with MIC values of up to 8 mg/ml are considered to possess at least

some degree of inhibitory effect, and any concentration exceeding this should not be

considered effective, according to Fabry et al. [180]. The majority of the crude bark

and leaf extracts of the six Canarium patentinervium Miq. exhibited moderate to

relatively good activity against Gram-positive and Gram-negative pathogens with the

MIC values ranging from 0.25 mg/ml to 8.00 mg/ml. Only the hexane extract of

leaves exhibiting weak antimicrobial activity. All active extracts displayed

concentration-dependent antimicrobial activity. Aligiannis et al. [181] proposed a

classification system based on MIC results obtained for plant materials, which was

consequently described and implemented by Duarte et al. [182].

Strong microbial inhibitors possessed MIC values of equal or lower than 0.50 mg/ml;

a clear indication that the MIC value of 0.25 mg/ml obtained for ethanol extracts (leaf

and bark) against MRSA Staphylococcus aureus (clinical strain) indicates exceptional

antimicrobial activity. The ethanol extract of leaves also resulted with the MIC of

0.50 mg/ml against MSSA (both strains) and MRSA (ATCC 43300). Coagulase-

negative Staphylococcus aureus: oxacillin resistant and sensitive strains were both

sensitive against both ethanol extracts with an MIC of 0.5 mg/ml for the bark extracts

and MIC range of 0.5 mg/ml to 1.00 mg/ml for the leaves. Staphylococcus aureus is a

Gram-positive bacteria that can cause a range of illnesses, from minor skin infections,

192

such as pimples, impetigo, boils, celluliti, folliculitis, carbuncles, scalded skin

syndrome and abscesses to life threatening diseases such as pneumonia, meningitis,

osteomyelitis, endocarditis, toxic shock syndrome, bacteremia and sepsis. Its

incidence ranges from skin, soft tissue, respiratory, bone, joint, endovascular to

wound infections [183]. MRSA is one of the greatly feared strains of Staphylococcus

aureus ZKLFKKDYHEHFRPHUHVLVWDQWWRPRVWȕ-lactam antibiotics and mostly hospital-

accquired. Coagulase-negative Staphylococcus aureus are differentiated by their

inability to produce free coagulase resulting in their inability to clot blood plasma

[184].

The ethanol extracts (bark and leaves) were bactericidal against Bacillus cereus with a

MIC of 0.50 mg/ml and MBC of 1.00 mg/ml. Bacillus cereus is a causative agent of

both gastrointestinal infections (diarrheal and emetic type of food poisoning) and non-

gastrointestinal infections (post traumatic wound and burn infections. ophthalmic

infections, endocarditis, postoperative meningitis and urinary tract infections) [185].

The ethanol extracts were also bactericidal against Gram-negative Klebsiella sp and

Klebsiella pneumoniae ESBL with MIC of 0.50 mg/ml and MBC of 2.00 mg/ml.

Klebsiella is well known to most clinicians as a cause of community-acquired

bacterial pneumonia, occurring particularly in chronic alcoholics which have a high

fatality rate if untreated. Nosocomial Klebsiella infections are caused mainly by

Klebsiella pneumoniae, the medically most important species of the genus. Due to the

extensive spread of antibiotic-resistant strains, especially of extended-spectrum b-

lactamase (ESBL)-producing strains, there has been renewed interest in Klebsiella

infections. Since ESBL production frequently is accompanied by multiresistance to

antibiotics, therapeutic options become limited [186].

193

The points above will make the discovery of an antimicrobial agent as effective as

Canarium patentinervium Miq. against these pathogens highly significant. The

ethanol extract of leaves displayed good microbial inhibition action against all

microbs tested except for Pseudomonas aeruginosa and Candida parapsilopsis with

moderate inhibition but bactericidal action with the MIC of 1.00 mg/ml and 2.00

mg/ml respectively. Pseudomonas aeruginosa is a Gram-negative bacterium and an

opportunistic, nosocomial pathogen in immunocompromised individuals. It typically

infects the pulmonary tract, urinary tract, burns, wounds and blood infections [187].

Moderate microbial inhibitors are described by Aligiannis et al. [181] as those plant

extracts with MIC values ranging between 0.60 mg/ml and 1.50 mg/ml. Amongst the

extracts investigated in the present study, hexane extracts of the barks seemed to have

most of the moderate inhibitors with an interesting bactericidal action against MRSA

ATCC 43300 strain (MIC of 1.00 mg/ml and MBC of 4.00 mg/ml). Weak microbial

inhibitors are classified as those agents with MIC values of between 1.60 mg/ml and

8.00 mg/ml [181]. None of the extracts in the present study yielded MIC values in

excess of 8.00 mg/ml against Gram-positive bacteria except with hexane extract of

leaves. The results obtained against the Gram-positive and Gram-negative bacteria

thus support the traditional use of Canarium patentinervium Miq. in wound

healing[164] and hold potential in the treatment of colds, wound healing and as an

antiseptic.

In general, the Gram-negative bacteria displayed the least sensitivity towards the

extracts, and most of the plant extracts (except ethanol extract of barks) exhibited

poor and unvaried activity against Esherichia coli, indicating the resistance of this

194

bacterium to the plant extracts. This was to be expected, as Gram-negative bacteria

offer a much more complex barrier system against permeation of foreign substances

(in this case, the antimicrobial agent). This is attributed to the specialised cell wall

structure and especially the presence of the outer envelope resulting in the

impermeability of these micro-organisms to biocides and antibiotics, and at times,

resulting in regulation and prevention of their passage to the target region [188].

Resistance to the plant extracts is, thus, exhibited to a far greater extent by the Gram-

negative bacteria than by Gram-positive bacteria [188]. The lipophilic or hydrophilic

nature of compounds also plays a role in the activity, or lack thereof, against the

micro-organisms. Compounds considered to be more effective against Gram-negative

bacteria are considerably less lipophilic. This is as a result of the structure of the

Gram-negative cell wall which also has a higher lipid content [189]. This could

explain why in this study the ethanol extracts which contain more hydrophilic

compounds showed more inhibition against Gram-negative bacteria such as

Esherichia coli and Klebsiella species.

The ethanol extracts and hexane extract of barks also showed inhibition against

Candida parapsilopsis with a cidal and static action respectively. Both ethanol

extracts had MIC of 2.00 mg/ml (MBC of 8.00 mg/ml) and hexane extract of barks

had an MIC of 8.00 mg/ml (MBC >16.00 mg/ml). More than 80% of all fungal

bloodstream infections are a direct result from the Candida species and since the

eighties there has been an explosive rise in the rate of Candidal infections. Over the

past decade, the incidence of Candida parapsilosis has dramatically increased. In fact,

reports indicate that Candida parapsilosis is often the second most commonly isolated

195

Candida species from blood cultures and Candida parapsilosis even outranks

Candida albicans in some European, Asian, and South American hospitals [190].

The ethanol extracts have shown to have polyphenols such as tannins and flavonoids

from the phytochemical analysis (chapter 2). Polyphenols, such as tannins and

flavonoids, have important antibacterial activity [191]. The antimicrobial activity of

flavonoids is due to their ability to complex with extracellular and soluble protein and

to complex with bacterial cell wall while that of tannins may be related to their ability

to inactivate microbial adhesions, enzymes and cell envelop proteins [163]. Flavones

are phenolic structures containing one carbonyl group (as opposed to the two

carbonyls in quinones). The addition of a 3-hydroxyl group yields a flavonol.

Flavonoids are also hydroxylated phenolic substances but occur as a C6-C3 unit

linked to an aromatic ring. Their activity is probably due to their ability to complex

with extracellular and soluble proteins and to complex with bacterial cell walls. More

lipophilic flavonoids may also disrupt microbial membranes [192].

Tannin is a general descriptive name for a group of polymeric phenolic substances

capable of tanning leather or precipitating gelatin from solution, a property known as

astringency. They are found in almost every plant part: bark, wood, leaves, fruits and

roots. Tannins may be formed by condensations of flavan derivatives or by the

polymerization of quinine units [191]. Many human physiological activities, such as

stimulation of phagocytic cell, host mediated tumour activity and a wide range of anti-

infective actions, have been assigned to tannins. Their mode of antimicrobial action as

described for quinone, may be related to their ability to inactivate microbial

adhesions, enzymes, cell envelope, transport-proteins etc [191]. They also form a

196

complex with polysaccharide. According to a number of studies, tannins can also be

toxic to filamentous fungi, yeasts and bacteria [192]. However, it is important to note

that, if tannins were solely responsible for the activity presented by these results, this

activity would be observed against all organisms and would not be limited to Gram-

positive bacteria or yeasts. The current hypothesis is thus that tannins are at least

partially responsible for the antibiotic activity. Coumarins have also demonstrated

antimicrobial activity [163].


$OOH[WUDFWVZLWKJRRGLQKLELWRUV0,&YDOXHPJPOwas tested for the time-kill

efficacy. All extracts displayed killing (bactericidal) action except the ethanol extracts

(leaves and barks) against Enterococcus faecalis which was only bacteriostatic

(Figure 7.17 & 7.18) and ethanol extract of barks that was bacteriostatic against

coagulase-negative Staphylococcus aureus: oxacillin resistant (Figure 7.14). The other

extracts bactericidal effect was achieved in the concentration of 2.00 to 4.00 mg/ml.

Killing rate was observed at varying times from range of 1 hr to 10 hrs all resulting in

complete kill.

7.5 Conclusion

All crude extracts of Canarium patentinervium Miq. under investigation exhibited

exceptional concentration-dependent antimicrobial activity against both Gram-

positive and Gram-negative bacteria as well as against yeasts. The most promising

197

activity was displayed against Gram-positive bacteria Staphylococcus aureus, MRSA,

Bacillus cereus and Gram-negative bacteria and Pseudomonas aeruginosa, Klebsiella

sp, Klebsiella pneumoniae ESBL and yeast Candida parapsilopsis. This serves as a

clear indication of the potential of these extracts for further chemical studies as

antimicrobial agents. The antimicrobial activity exhibited by the bark extracts and

ethanol extract of leaves against MSSA and MRSA indicates exceptional

antimicrobial activity. All the tested bacterias and yeast were susceptible to either

bark or leaf extracts.

In general, the Gram-negative bacteria Escherichia coli displayed the least sensitivity

towards the extracts, being inhibited only by the ethanol extract of the bark indicating

the resistance of this bacterium to the rest of the plant extracts. The activity against

Klebsiella pneumonia ESBL and Klebsiella sp. was, however, far more promising

with variability amongst the plant extracts. It is at this stage important to note that the

failure of a plant extract to demonstrate in vitro activity during the general screening

process does not necessarily imply a total absence of inherent medicinal value. The

possible presence of synergistic interactions between the different plant constituents

in crude preparations may result in activities that are not exhibited by isolated

compounds, and should not be excluded.

198

CHAPTER 8

ANTIPARASITIC ACTIVITY

8.1 Introduction

8.1.1 Parasitic infection: causative factor and treatment

Parasitic infection is one of a major health problem in the developing world. Twenty-

five percent of the world's population could be suffering from parasitic infection

Highest prevalence is in underdeveloped agricultural and rural areas in the tropical

and subtropical regions. In some areas incidence may reach 90 % of the population.

During the evolution of humans a broad set of parasites have evolved, that use us as a

host organism [193]. Usually a parasite will not kill its host (at least not immediately),

as this would be an evolutionary dead end for a parasite. However, most parasites are

either unpleasant for us (think of lice and fleas) or weaken our health (most internal

parasites). However, some parasitic infections, such as malaria, trypanosomiasis or

Chagas can be deadly if the patients are not treated with adequate therapeutics.

Because humans usually live in close proximity and often without good hygienic

conditions the transmission of parasites within a human population is often facilitated

[194].

Many parasitic infections are the cause of tropical diseases, such as malaria,

trypanosomiasis, leishmaniasis, Chagas disease, schistosomiasis, onchocerciasis,

lymphatic filariasis, and helminthiases. Parasites are responsible for probably more

199

than 1±2 billion infections, which lead to several million deaths every year [195]. An

overview on important endoparasites under this study, their vectors, distribution, and

disease symptoms is given in Table 8.1.

Table 8.1: Human endoparasites in study

Parasite Disease (estimated number of infections)

Vector (hosts); route of transmission

Distribution Symptoms

Trypanosomatida

Leishmania donovani

Visceral leishmaniasis (kala-azar) (15 million)

Flies (Phlebotomus, Lutzomyia); bites

N.Africa, Eurasia, S America

Enlargement of liver and spleen, fever, dermal lesions, dermal nodules

Amoebida

Entamoeba histolytica and other species

Amoebiasis (70000 death/year)

Infection from contaminated water or food

Worldwide Dysentery, destruction of intestinal tissues, fever, liver and lung abscess

Diplomonadida

Giardia lamblia Giardiasis (2 million infections/ year in USA)

Infection from contaminated water

Worldwide Infection of duodenal and jejunal mucosa; diarrhoea, fever

Leishmaniasis is a group of disease, caused by Leishmania species. The disease is

considered as a major public health problem in 82 countries in the world causing

morbidity and mortality [196]. Different modes of treatment are used in the treatment

of cutaneous leishmaniasis. Pentavalent antimonial compounds are the first line

treatment but generally are toxic so several significant advances in the chemotherapy

of the leishmaniasis have occurred in the last 10 years [193]. Leishmaniasis is caused

by protozoan parasites of the genus Leishmania which invade macrophages of host

200

organisms. A distinction is made between cutaneous, mucocutaneous, visceral and

diffuse leishmaniasis, of which visceral leishmaniasis is a fatal disease causing

approximately 60,000 death per year [197]. Leishmaniasis and HIV infections often

co-occur and these patients usually have a poor prognosis. Patients are treated with

the synthetic drugs stibogluconate, meglumine and pentamidine (developed 70 years

ago), which have severe side effects and fail to work in North Bihar (India)

[198][199]. Also the macrolide antibiotic amphotericin B has been employed, which

can also be toxic for patients. New developments include the anticancer drug

miltefosine, the aminoglycoside antibiotic paronomycin, and the 8-aminoquinoline

sitamaquine [197]. Among natural products, berberine (which occurs in many TCM

plants) had promising anti-leishmanial activities [198][199].

Entamoeba histolytica and Giardia intestinalis are parasite of large and small

intestine of human, respectively. Although infection with E. histolytica is

asymptomatic in 90% of cases, about 50 million people are estimated to suffer from

the symptoms of amoebiasis such as haemorrhagic colitis and amoebic liver abscess.

These infections result in 50,000 to 100,000 deaths annually [200]. Giardia

intestinalis (Synonyms: Giardia duodenalis, Giardia lamblia) is one of the most

common universal pathogenic intestinal protozoan parasites of humans [201]. It is

also widely spread in various animals such as dogs [202], cats [203] and cattle [204].

It is frequently reported to be a significant cause of diarrhea in people traveling to

WURSLFDOFRXQWULHVWUDYHOHU¶VGLDUUKHD$OWKRXJKWKHPDMRULW\RILQIHFWHGKXPDQVVKHG

Giardia cysts without any symptoms, the major clinical signs of giardiasis in humans

include acute or chronic diarrhea, nutritional disorders and weight loss. Recent studies

have shown that Giardia genotypes may be shared between human and numerous

201

other species of animal [205][206] indicating transmission between humans and

animal vectors may occur, possibly through exposure to contaminated water.

Metronidazole, the current drug of choice, can cause mutagenicity in bacteria [207]

and is carcinogenic in rodents [208][209]. It also possesses undesirable side effects

and failures in treatment have been reported [210][211]. We therefore plan to evaluate

the in vitro activity of Canarium patentinervium Miq., against Giardia intestinalis in

the search for an alternative drug suitable for use in preventing and treating cases of

diarrhoea.

8.1.2 Natural products and parasitic defence

We know that humans have used medicinal plants for several thousands of years to

treat illness and health disorders [212]. Medicinal chemists have synthesized a

number of drugs which can be used against many but by far not all endoparasites. A

major problem is that many of these drugs were developed many years ago and some

parasitic strains have become resistant to them. The development of new antiparasitic

drugs has not been much of a priority for the pharmaceutical industry because many

of the parasitic diseases occur in poor countries where the populations cannot afford

to pay a high price for the drugs. Thus an investment in drug development against

parasitic diseases is a risky affair. An alternative to synthetic drugs is the search for

anti-parasitic plant extracts or secondary metabolites derived from them. Natural

products still play an important role in therapy: between 1981 and 2006, 1,184 new

drugs were registered of which 28% were natural products or their derivatives.

Another 24% of the new drugs had pharmacophores (i.e., functional groups with

202

pharmacological activity) derived from natural products [213]. A good starting point

to find antiparasitic natural products would be traditional medicinal plants, such as

those known from Asia, Africa or America [212] that have been employed to treat

infections.


8.2.1 Principle of method

The antiprotozoal activity of the compounds was perform according to the standard

methods as described by Sawangjaroen et al. [214][200]. The antileishmanial activity

of the compounds was perform according to the standard methods as described by

Mosmann [215].

8.2.2 Protocol

8.2.2.1 Antiparasitic assay against Giardia intestinalis and Entamoeba histolytica

8.2.2.1.1 Test organisms

Giardia intestinalis

A local Thai strain of Giardia intestinalis, originally described by Siripanth et al.

[216] was used throughout this experiment.

203

Entamoeba histolytica

Entamoeba histolytica strain HM1: IMSS, purchase from ATCC was used throughout

this experiment.

8.2.2.1.2 Cultivation of organisms

Both Giardia intestinalis and Entamoeba histolytica were cultured axenically in screw

capped tubes at 37 oC, under anaerobic condition, on YI medium [217] supplemented

with 10% heat-inactivated horse serum. Subculture was performed every 48 hr. For

assays, trophozoites were harvested by chilling the tube on ice for 15 min to detach

the monolayer and centrifuged at 300 g for 5 min. The supernatant was decanted and

cells were re-suspended in fresh medium. The numbers of viable cells were calculated

using a haemocytometer and 0.4 % (w/v) trypan blue. The criteria used for viability

were motility and dye exclusion.

8.2.2.1.3 Antiprotozoal assay

Briefly, trophozoites, 2x105 cells/ml, was incubated, in triplicate, in the presence of

serial two fold dilutions of plant extracts that ranged from 31.25 to 500 Pg/ml for

crude extract in 96 well tissue culture plates (200 Pl/well). Metronidazole and

complete medium with added DMSO will be used as negative and positive control,

respectively. After 24 hr of incubation at 37 oC under anaerobic conditions, the

trophozoites from each well will examine with an inverted microscope. The

appearance of trophozoites will score and presented as score values from 1 to 4 with 1

showing the most inhibition of growth and 4 showing no inhibition, according to

204

Upcroft and Upcroft [218]. The minimum inhibitory concentration (MIC) will be

recorded (the lowest concentration at which >90% of the trophozoites rounded up).

Each concentration was tested in triplicates and at least three experiments were

performed on separate occasions.

8.2.2.2 Antiparasitic activity against Leishmania donovani promastigotes

Antileishmanial activity of tested plant extract, against Leishmania donovani (strain

MHOM/IN/1983/AG83) promastigotes, was evaluated by a quantitative colorimetric

assay using MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide;

Sisco Research Laboratory, Mumbai, India]. Amphotericin B (Sigma-Aldrich Co.,

MO, USA; IC50 =0.4±0.1µM) was used as the positive control in all the experiments.

Promastigotes (5×105 cells/ml; 300 µl), were treated with and without tested samples

at concentrations of 100 and 500 µg/mL, and incubated at 22 ± 2 oC. After 72 hr, cells

were harvested, and re-suspended in PBS (500 µl) containing MTT (0.3 mg/ml).

Purple formazan crystals were dissolved in DMSO and the optical density (O.D.) was

measured at 570 nm in an ELISA reader (BIO-RAD; model 680, USA). The number

of viable cells was directly proportional to the amount of formazan produced through

the reduction of yellow MTT by the dehydrogenase enzymes present in the inner

mitochondrial membrane of the living cells.

The percentage of growth inhibition was calculated as follows:

% inhibition = [(O.D. of untreated control - O.D. of treated set)/ O.D. of untreated

control] x 100.

205


The IC50 values (concentration of drug which inhibited at least 50% cell growth) for

each compounds were determined from respective dose-responsive percentage

inhibition curves with the help of Microsoft Excel and data were reported as mean and

SD values obtained from a minimum of three determinations. Graph was plotted with

SD and 95% confidence interval.

8.3 Results

The result of the in vitro effect of extracts on Giardia intestinalis and Entamoeba

histolytica was summarized in Table 8.2. It was found that the ethanol extracts were

both active against growth of Giardia intestinalis in vitro with MIC 500 µg/ml (Table

8.2). The MIC of metronidazole was 2.5 µg/ml and the remaining extracts showed no

activity (MIC >500µg/ml for crude extract). The appearance of trophozoites in

negative, positive and with exposure to LE under microscope is shown in Figure 8.1.

The result of the in vitro effect of extracts on Leishmania donovani was summarized

in Table 8.3. The potency of extracts against Leishmania donovani was LH > BH >

BC > LC. Both ethanol extracts of leaves and barks had IC50 values of above 500

µg/ml. Hexane extract displayed lowest IC50 value of 257.40±0.30 µg/ml. Figure 8.2

shows the inhibition of the hexane and chloroform extracts.

206

8.4 Discussion

Parasites are eukaryotes and therefore share most molecular and biochemical

properties with their eukaryotic hosts, making it often difficult to find antiparasitic

drugs which are both effective and non-toxic for humans. This limitation always has

to be kept in mind when discussing the numerous findings that some drug or extract

from a medicinal plant is active against parasites in vitro. In order to be medicinally

useful, such a drug must have bioavailability and should not intoxicate the patient. A

first guidance is the determination of a selectivity index (SI) which compares the

cytotoxicity of a drug against a parasite and a library of human cells [194].

Most of the antiparasitic properties of extracts and isolated natural products have been

tested in vitro only. Translation of the in vitro research results into in vivo trials is

urgently required. Furthermore, even if animal experiments were successful, we

would need clinical trials of the new compounds alone or in combination with

established parasiticidal drugs to prove their efficacy and safety. These developments

are costly and it is presently difficult to attract the pharmaceutical industries into these

fields for various reasons. TCM and other traditional medicine systems employ

several thousand of medicinal plants; some of which have known antiparasitic

properties.

207

Table 8.2: MIC values of Canarium patentinervium Miq. incubated for 24 hr with Giardia intestinalis and Entamoeba histolytica growing in vitro.

Sample MIC (µg/ml)

Giardia intestinalis Entamoeba histolytica

LH >500 >500

LC >500 >500

LE 500 >500

BH >500 >500

BC >500 >500

BE 500 >500

Metronidazole 2.5 2.5

LH: leaf hexane extract, LC: leaf chloroform extract, LE: leaf ethanol extract, BH: bark hexane extract, BC: bark chloroform extract, BE: bark ethanol extract. Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same capital letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

208

Figure 8.1: Appearance of trophozoites under microscope in negative control, positive control and with LE

Negative control of G. intestinalis

Negative control of E. histolytica

Positive control: 10 Pg/ml of metronidazole ;cell deathͿ

Positive control: 10 Pg/ml of metronidazole;cell deathͿ

209

Table 8.3: Antiparasitic values of Canarium patentinervium Miq. against Leishmania donovani (AG 83) promastigotes

Sample IC50 (µg/ml)

LH 257.40±0.30

LC 457.70±0.25

LE >500

BH 284.20±0.40

BC 359.90±0.20

BE >500

LH: leaf hexane extract, LC: leaf chloroform extract, LE: leaf ethanol extract, BH: bark hexane extract, BC: bark chloroform extract, BE: bark ethanol extract. Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same capital letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

210

0 200 400 600

0

20

40

60

80BH

BC

LH

LC

% in

hibi

tion

agai

nst

L.

do

no

van

i


Figure 8.2: MTT assay of Canarium patentinervium Miq. against Leishmania donovani.

211

They offer a unique opportunity to identify natural products which could be

potentially used to treat parasitic infections. The success story of artemisinin from

Artemisia annua [213][219] can probably be repeated. A number of medicinal plants

and secondary metabolites isolated from them have been screened for anti-

trypanosomal activity [220]. Active natural products include several groups of

alkaloids, phenolics, saponins, cardiac glycosides, other terpenoids, and

polyacetylenes (common in Apiaceae, Asteraceae and Araliaceae). Although some

natural products are active in the submicromolar range and show good selectivity,

only few have been studied in vivo in an animal model.

In the present study, six different extracts of Canarium patentinervium Miq.

(Burseraceae) leaves and barks were screened for their in vitro antiparasitic activities,

among the different extracts tested, the hexane extract of leaves showed moderate

antileshmanial activity with IC50 values of 257.40±0.30 µg/ml. This could be due to

essential oils present in the hexane extracts as shown previously in the family of

Burseraceae [221]. Only ethanol extracts showed activity against Giardia intestinalis

and Entamoeba histolytica at concentration of 500 µg/ml. Other extracts were inactive

against these two intestinal parasites.

Small lipophilic secondary metabolites, such as terpenoids or phenylpropanoids as

found in the essential oil of many plants (especially in Lamiaceae, Myrtaceae,

Rubiaceae, Apiaceae, Asteraceae, Lauraceae, Rutaceae, Burseraceae, Verbenaceae,

Pinaceae, Cupressaceae), can dissolve in biomembranes and disturb their fluidity and

the function of membrane proteins [221]. Therefore, many of the lipophilic mono- and

sesquiterpenes, phenylpropanoids and isothiocyanates (as present in Brassicaceae,

212

Tropaeoleaceae) have a certain degree of antimicrobial and antiparasitic properties

[222].

8.5 Conclusion

Plant substances continue to serve as a wellspring of drugs for the world population

and several plant-based drugs are in extensive clinical use. Preliminary antiparasitic

screening of six extracts of Canarium patentinervium Miq. showed that the ethanol

extract had promising growth inhibition on Giardia intestinalis and Entamoeba

histolytica. The hexane extract of leaves was active against Leshmanial donovani. The

results obtained suggest that further isolation studies can be performed on Canarium

patentinervium Miq to identify the active constituents responsible for the antiparasitic

activities.

213

CHAPTER 9

ANTICANCER ACTIVITY

9.1 Introduction

9.1.1 Carcinogenesis

Cancer, a cellular malignancy that results in the loss of normal cell-cycle control, such

as unregulated growth and the lack of differentiation, can develop in any tissue of any

organ, and at any time [223] .

Carcinogenesis is the transformation of a normal cell to a cancerous cell through

many stages, which occur over a number of years or even decades. The first stage of

carcinogenesis is the initiation stage, which involves the reaction between the

carcinogens and the DNA of the cells. Inhibiting this early stage of cancer is an

important strategy in cancer prevention or treatment. Promotion is the second stage

and may occur slowly over an extended period of time, ranging from several months

to years. Beneficial effects may arise from a change in lifestyle and diet, which may

result in the individual not developing cancer during his or her lifetime. The third

stage is the progressive stage, involving the spread of the cancer. It is evident that,

upon entering into this stage, preventative factors such as diet have less of an impact.

214

Despite the therapeutic advances made in understanding the processes involved in

carcinogenesis, cancer has become one of the most serious medical problems today.

The worldwide mortality rate increases annually, with more than seven million deaths

occurring per year. For this reason, cancer chemotherapy has become a major focus

area of research. Different lifestyles, risk factors (such as age, gender, race, genetic

disposition) and the exposure to different environmental carcinogens, lead to the

varying patterns of cancer incidence [223]. At least 35% of all cancers worldwide

result from an incorrect diet, and in the case of colon cancer, diet may account for

80% of these cases [224]. One of the most important contributions to the development

of cancer is the oxidative damage to DNA [225]. Permanent genetic alterations may

occur in those cells where DNA is damaged and where division of this DNA occurs

before it can be repaired. These cells may begin to divide more rapidly and result in

carcinogenesis [224].

9.1.2 Natural products and carcinogenesis defence

³&KHPRSUHYHQWLRQ´ LV GHILQHG as a process to delay or prevent carcinogenesis in

humans through the ingestion of dietary or pharmaceutical agents. This also implies

the identification of chemical entities (specifically cytotoxic entities) that are effective

against a range of cancer cell lines, although less active or non-toxic against the

normal (healthy) cell population. The search for such anticancer agents from plant

VRXUFHV VWDUWHG LQ WKH ¶V DQG Slant products have proven to be an important

source of anticancer drugs [226]. This directly results from the biological and

chemical diversity of nature, which allows for the discovery of completely new

chemical classes of compounds.

215

The discovery and development of plant-derived compounds led to the first treatment

of cancer, specifically upon administration of these compounds in combination with

synthetic agents. At present more than 450 different compounds have been isolated

from active plants that have shown in vitro and/or in vivo antitumor activity. Virtually

every major class of natural chemical compound is represented in the list of active

constituents [227]. Of the 121 medications being prescribed for use in cancer

theraphy, 90 are sourced from plants. It was also determined that approximately 74%

of these discoveries were as a result of an investigation into the claims made by

folkloric tradition [228]. A number of natural products are used as chemoprotective

agents against commonly occurring cancers. Examples of these compounds used as

cytotoxic drugs are shown in Table 9.1.

A number of mechanisms exist by which phytochemicals aid in the prevention of

cancer. This preventative action most probably results from the additive or synergistic

effects of a number of phytochemicals, since cancer is a multi-step process [227].

Proposed mechanisms by which phytochemicals may prevent cancer include: (i) anti-

oxidant and free radical scavenging activity; (ii) antiproliferative activity; (iii) cell-

cycle arresting activity; (iv) induction of apoptosis; (v) activity as enzyme cofactors;

(vi) enzyme inhibition; (vii) gene regulation; (viii) activity as hepatic phase I enzyme

inducers, and (ix) activity as hepatic phase II enzyme inducers. Oxidative damage to

DNA, proteins and lipids, resulting from an increase in oxidative stress, is considered

to be one of the most important mechanisms contributing to the development of

cancer [227] .

216

The phytochemicals that most often appear to be protective against cancer are

curcumin, genistein, resveratrol, diallyl sulfide, (S)-allyl cystein, allicin, lycopene,

ellagic acid, ursolic acid, catechins, eugenol, isoeugenol, isoflavones, protease

inhibitors, saponins, phytosterols, vitamin C, lutein, folic acid, beta carotene, vitamin

E and flavonoids, to name but a few [224]. These phytochemicals suppress the

inflammatory processes that lead to transformation, hyperproliferation, and initiation

of carcinogenesis. The inhibitory influences of these phytochemicals may ultimately

suppress the final steps of carcinogenesis via angiogenesis and metastasis.

The present study was undertaken to screen the anticancer potential Canarium

patentinervium Miq. No anticancer studies have previously been reported on this

species to date.

217

Table 9.1: Cytotoxic drugs developed from plant sources.

Therapeutic agent Plant source Mechanism of action

Treatment of cancer type

Reference

Vincristine, vinblastine

Catharanthus roseus

Inhibition of tubulin polymerisation

+RGJNLQ¶VGLVHDVH [229]

Etoposide, teniposide Podophyllum peltatum

Inhibition of topoisomerase II

Testicular cancer, and small cell lung carcinoma, leukaemias, lymphomas

[229] [230] [231]

Paclitaxel, docetaxel Taxus brevifolia

Promotion of tubulin stabilization

Ovarian and breast carcinoma

[229]

Irinotecan, topotecan, 9-aminocampothecin, 9-nitrocamptothecin

Camptotheca acuminata

Inhibition of topoisomerase I

Advanced colorectal cancer, also active in lung, cervix and ovarian cancer

[230]

Homoharingtonine Harringtonia cephalotaxus

Inhibition of DNA polymerase

Various leukaemias [229]

4-Ipomeanol Ipomoea batatas

Cytochrome P-450- mediated conversion into DNA-binding metabolites

Lung cancer [229]

Elliptinium Bleekeria vitensis

Inhibition of topoisomerase II

Advanced breast cancer

[229]

Flavopiridol Amoora rohituka, Dysoxylum binectariferum

Inhibition of cyclin- dependent kinases

Encouraging results noted in a variety of solid and haematological malignancies, in patients with colorectal, prostate, lung, renal carcinoma, non-+RGJNLQ¶Vlymphoma and chronic lymphocytic leukaemia

[229]

218


9.2.1 Principle of the MTT assay

MTT assay is dependent on the reduction of the tetrazolium salt MTT (3-(4,5-

dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide) by the mitochondrial

dehydrogenase of viable cells to form a blue formazan product. The assay measures

cell respiration and the amount of formazan produced is proportional to the number of

living cells present in culture which will result in lower optical density (OD) [232].

Biological in vitro assay was determined as follows [233].

9.2.2 Protocol

9.2.2.1 Cell lines and cell culture

Human derived cell lines [HCT 116,colon cancer cell line and MCF-7 (ER+), MDA

468 (ER-) breast carcinoma] were routinely cultivated at 37 °C in an atmosphere of 5

% CO2 in RPMI 1640 medium supplemented with 2 mM L-glutamine and 10 % fetal

calf serum and subcultured twice weekly to maintain continuous logarithmic growth.

Cells were seeded into 96-well microtiter plates at a density of 3-5 × 103 per well and

allowed 24 hr to adhere before extracts were introduced (final concentration 200

µg/ml to 1 µg/ml).

219

9.2.2.2 Preparation of plant samples

Extracts were prepared as 400 µg/ml top stock solutions, dissolved in DMSO, and

stored at 4 oC, protected from light for a maximum period of 4 weeks. Serial sample

dilutions were prepared in medium immediately prior to each assay.

9.2.2.3 The MTT assay

At the time of extract addition and following 72 hr exposure, MTT (3-(4,5-

dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide) was added to each well.

Incubation at 37 °C for 4 hr allowed reduction of MTT by viable cells to an insoluble

formazan product. Well contents were aspirated and formazan solubilized by addition

of DMSO:glycine buffer (pH 10.5) (4:1). Absorbance was read on an Anthos Labtec

System plate reader at 550 nm as a measure of cell viability, thus cell growth or

extract toxicity was determined. Doxorubicin was used as positive control.


Concentration±response curves were calculated using Microsoft Excel and data were

reported as mean and SD values obtained from a minimum of three determinations.

Graph was plotted with SD and 95% confidence interval.

220

9.3 Results

A range of extracts were evaluated in MTT assays following a 3-day exposure against

a panel of two human breast cancer cell lines, MCF-7 (ER+) and MDA 468 (ER-),

and a colon cancer cell line, HCT 116. In general, the breast cancer cell lines were

more sensitive to the extracts, and the MDA 468 line was the most sensitive of the

three lines (Table 9.2) The best growth inhibition was observed with the chloroform

and ethanol extract of barks, GI50 values of 23.44 µg/ml and 34.40 µg/ml respectively

(Figure 9.1-9.3).

9.4 Discussion

In the present study, six different extracts of Canarium patentinervium Miq.

(Burseraceae) leaves and barks were screened for their in vitro antitumor activities.

Among the different extracts tested, the chloroform and ethanol extract of barks

showed good antitumor activities with GI50 values of 23.44±0.05 µg/ml and

34.40±0.21 µg/ml respectively. The most susceptible cell lines were found to be the

breast cancer cell line, MDA 468.

It is known that nature is able to produce a wide variety of chemical entities of novel

structure. Many of the new and novel compounds isolated from natural sources might

otherwise have never been discovered, especially those of considerable complexity

requiring the development of methods for the creation of new ring systems.

221

Table 9.2: Growth inhibition of human cancer cell lines by crude plant extracts of Canarium patentinervium Miq.

Extracts Cell line/ GI50 (µg/ml)

MDA 468 HCT 116 MCF-7

LH >100 >100 >100

BH >100 >100 >100

LC >100 >100 >100

BC 23.44±0.05 62.46±0.23 64.50±0.27

LE 61.48±0.23 97.18±0.08 85.40±0.18

BE 34.4±0.21 46.53±0.12 39.06±0.16

Doxorubicin 0.06±0.12 0.70±0.18 0.34±0.26

LH: leaf hexane extract, LC: leaf chloroform extract, LE: leaf ethanol extract, BH: bark hexane extract, BC: bark chloroform extract, BE: bark ethanol xtract Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

222

Figure 9.1a: Effects of extract of leaves against MDA 468 growth

0

0.2

0.4

0.6

0.8

1

1.2

control 0.5 µg/ml 1 5 10 50 100

OD

550 n

m

concentration (µg/ml)

chloroform

ethanol

hexane

T zero

223

Figure 9.1b: Effects of extract of barks against MDA 468 growth

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

control 0.5 µg/ml 1 5 10 50 100

OD

550 n

m


chloroform

ethanol

hexane

T zero

224

Figure 9.2a: Effects of extract of leaves against MCF-7 growth

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

control 0.5 µg/ml 1 5 10 50 100

OD

550 n

m


chloroform

ethanol

hexane

T zero

225

Figure 9.2b: Effects of extract of barks against MCF-7 growth

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

control 0.5 µg/ml 1 5 10 50 100

OD

550 n

m


chloroform

ethanol

hexane

T zero

226

Figure 9.3a: Effects of extract of leaves against HCT 116 growth

0

0.5

1

1.5

2

2.5

control 0.5 µg/ml 1 5 10 50 100

OD

550 n

m


chloroform

ethanol

hexane

T zero

227

Figure 9.3b: Effects of extract of barks against HCT 116 growth

-0.5

0

0.5

1

1.5

2

2.5

control 0.5 µg/ml 1 5 10 50 100

OD

550 n

m


chloroform

ethanol

hexane

T zero

228

Natural products appeared to be a promising source for new types of compounds with

antitumor activity [234].

9.5 Conclusion

Plant substances continue to serve as a wellspring of drugs for the world population

and several plant-based drugs are in extensive clinical use [3]. Preliminary anticancer

screening of six extracts of Canarium patentinervium Miq. showed that the

chloroform and ethanol extract of the barks had promising effect on the cell viability

of breast cancer cell line MDA 468. The results obtained suggest that further isolation

studies can be performed on Canarium patentinervium Miq to identify the active

constituents responsible for the anticancer activities.

229

CHAPTER 10

ISOLATION AND IDENTIFICATION OF COMPOUNDS

10.1 Introduction

10.1.1 Secondary metabolites

Medicinal plants have formed the basis of health care since earliest times of humanity

and are still being widely used. The clinical, pharmaceutical and economic value

continues to grow, varying between countries. Chemodiversity in plants has proven to

be important in pharmacological research and drug development, not only for the

isolation of bio-active compounds used directly as therapeutic agents, but also as

leads to the synthesis of drugs or as models for pharmacologically active compounds

[235]. The rapid identification of these bio-active compounds, however, is critical if

this tool of drug discovery is to compete with developments in technology. Plant

preparations are distinguished from chemical drugs due by their complexity of

mixtures containing large numbers of bio-active compounds or secondary metabolites.

This brings about the challenge of drug discovery from natural sources [236].

Plants produce primary and secondary metabolites which encompass a wide array of

functions. Primary metabolites, which include amino acids, simple sugars, nucleic

acids, and lipids, are compounds that are necessary for cellular processes [237].

Secondary metabolites include compounds produced in response to stress, such as the

case when acting as a deterrent against herbivores. Plants can manufacture many

230

different types of secondary metabolites, which have been subsequently exploited by

humans for their beneficial role in a diverse array of applications [237]. Although

secondary metabolites from natural products have formed the basis of medicines, the

presence of these compounds in the biochemistry of the plant is very often difficult to

justify. It has been suggested that these compounds may have been synthesized by the

plant as part of the defense system of the plant, e.g. plants are known to produce

phytoalexins as a response to attack by bacteria and fungi [236]. The presence of

highly toxic natural products has also been highlighted in some animals namely the

Amazonian frogs so as to deter predation by other animals [236].

Secondary metabolites are organic molecules that are not involved in the normal

growth and development of an organism. While primary metabolites have a key role

in survival of the species, playing an active function in the photosynthesis and

respiration, the absence of secondary metabolites does not result in immediate death

but rather in long-term LPSDLUPHQWRI WKHRUJDQLVP¶VVXUYLYDELOLW\RIWHQSOD\LQJDQ

important role in plant defense [238]. Secondary metabolites are frequently produced

at highest levels during a transition from active growth to stationary phase [238]. The

producer organism can grow in the absence of their synthesis, suggesting that

secondary metabolism is not essential, at least for short term survival. A second view

proposes that the genes involved in secondary metabolism provide DµµJHQHWLFSOD\LQJ

ILHOG´ WKDW DOORZV PXWDWLRQ DQG QDWXUDO VHOHFWLRQ WR IL[ QHZ EHQHILFLDO traits via

evolution. A third view characterizes secondary metabolism as an integral part of

cellular metabolism and biology; it relies on primary metabolism to supply the

required enzymes, energy, substrates and cellular machinery and contributes to the

long term survival of the producer [238].

231

Different classes of these compounds are often associated to a narrow set of species

within a phylogenetic group and constitute the bioactive compound in several

medicinal, aromatic, colorant, and spice plants and/or functional foods. These

compounds are an extremely diverse group of natural products synthesized by plants,

fungi, bacteria, algae, and animals [239]. A simple classification of secondary

metabolites includes three main groups: terpenes (such as plant volatiles, cardiac

glycosides, carotenoids and sterols) constituting about 25,000 types, phenolics (such

as phenolic acids, coumarins, lignans, stilbenes, flavonoids, tannins and lignin)

constituting about 8,000 types and nitrogen containing compounds (such as alkaloids

and glucosinolates) constituting about 12,000 types [240]. Whatever the reasons for

the presence of these compounds in nature, they provide an invaluable resource that

have been used to find new drug molecules [241]. A number of traditional separation

techniques with various solvent systems and spray reagents, have been described as

having the ability to separate and identify secondary metabolites [48][238].

When an active extract has been identified, the first task is the identification of the

bio-active phytocompounds. The coupling of preliminary bioassay screening on crude

extracts and chromatographic methods such as column chromatography, preparative

thin layer chromatography (PTLC), high performance liquid chromatography

(HPLC), mass spectrometry (MS) and nuclear magnetic resonance (NMR)

spectroscopy, eventually leading to final bioassay screening, are important for the

acquisition of biologically active compounds.. The successful isolation of compounds

from plant materials largely depends on the type of solvent used for extraction [242].

The extract is separated into individual components and the biological activity of each

fraction is determined until a pure active compound is obtained. The pure compound

232

is then identified using methods such as mass spectrometry and nuclear magnetic

resonance spectroscopy.

Research has demonstrated that natural products represent an unparalleled reservoir of

molecular diversity. The isolation and identification of bio-active phytocompounds in

medicinal plant extracts generally used by local population to treat diseases would

prove an immeasurable contribution to drug discovery and development [235]. This

would not only validate the traditional use of herbal remedies but also provide leads in

the search for new active principles. Investigations surrounding the constituents of the

indigenous Canarium patentinervium Miq. in general, have not been the focus of any

phytochemical study.

10.1.2 Isolation methods

Once the extract has been obtained, the activity within can be demonstrated by

bioassay methods using both the crude extract or by using the fractionated extracts.

Fractionation has the added advantage of getting to the biologically active material

faster. One of the simplest separation methods is partitioning which is a widely used

method as an initial extract purification step. A combination of solvents²miscible

and immiscible ones are used to separate the phytochemicals making up the extract.

This method relies on the ability or the components to be either soluble in water or in

the organic phase [241]. This was then followed by various methods of

chromatography to isolate the bioactive molecules.

233

10.1.3 Crude Fractionation

The objectives at this stage are to simplify the extract composition by dividing it into

groups of compounds sharing similar physicochemical characteristics and/or to

remove the bulk of unwanted materials and thus enrich the extract with respect to the

target compounds. Procedures commonly employed involve solvent partitioning,

defatting, and desalting. The procedure used in this study is a modification of the

method developed by Kupchan [48]. It can be used for defatting and desalting as well.

Most of fats will go with the n-hexane fraction, while inorganic salts will go with the

aqueous one. The advantage of the method is total recovery of target compounds.

Drawbacks are the problems of emulsion formation, time ineffectiveness, and use of

large volumes of solvents.

10.1.4 Chromatography

µ&KURPDWRJUDSK\¶ LV WKH JHQHUDO WHUP IRU D YDULHW\ RI physico-chemical separation

techniques, all of which have in common the distribution of a component between a

mobile phase and a stationary phase. The stationary phase is fixed in the system and

the mobile phase which is a fluid, streams through the chromatographic system. The

various chromatographic techniques are sub-divided according to the physical state of

these two phases. The molecules of the analytes are distributed between the mobile

and the stationary phases. When present in the stationary phase, they are retained and

do not move through the system but when present in the mobile phase, they migrate

with a velocity equivalent to that of the mobile phase. Due to the different

distributions of the analytes, their mean residence time in the stationary phase differs,

234

resulting in a different 40 net migration velocity. This is the principle of the

chromatographic system [243]. Chromatograhic techniques have been instrumental in

the separation of natural products.

10.1.4.1 Thin Layer Chromatography (TLC)

One of the fastest and most widely used chromatographic techniques is Thin Layer

Chromatography. TLC is an analytical technique that can be used to monitor reactions

and for the qualitative analysis of complex mixtures and for the identification of

unknown compounds. It is also important for determining the correct solvent system

with which to run a column chromatography. TLC is composed of two phases, a

mobile and a solid phase. The solid phase is a thin solid support that usually consists

of alumina or silica [48].. The mobile phase is a solvent that moves through capillary

action right through the solid phase. In general, the solid phase is usually polar while

the mobile solvent is non polar relative to the solid phase. The choice of the solvent

depends on the properties of compounds that you want to separate and the nature of

the solid support you are using (ie, is it silica or alumina). If some compounds cannot

be visualized with eyes UV light can be used and a variety of visualisation reagents

can be used [48].

The behaviour of a compound on a TLC is usually described in terms of its relative

mobility or retention factor (Rf) value. Rf is a unique value for each compound under

the same conditions. The Rf for a compound is a constant from one experiment to the

next only if the chromatography conditions (solvent system, adsorbent, thickness of

adsorbent, amount of material spotted and temperature) are also constant [244]. Since

235

these factors are difficult to keep constant from experiment to experiment, relative Rf

YDOXHV DUH JHQHUDOO\ FRQVLGHUHG ³5HODWLYH 5I´ PHDQV WKDW WKH YDOXHV DUH UHSRUWHG

relative to a standard. Based on the Rf values, the identity and characteristics of the

different compounds can be determined. More polar compounds will have smaller Rf

values since they will have a stronger affinity for the polar solid phase. This would

result in the compound not being carried very far along the TLC plate. Compounds

with larger Rf values interact strongly with the less polar mobile phase and thus, tend

to be non polar themselves [238].

PTLC is used for analytical separations of larger quantities of materials. Because

sample mass loading capacity is proportional to the square root of the sorbent layer

thickness, thicker layers are used (>250 ȝPXSWRPP:KHQDELQGHULVUHTXLUHG

the softer inorganic binders (like calcium sulphate) are used so the sample bands can

be easily removed. The compounds to be separated are often applied as long streaks,

developed and then recovered by scraping the adsorbent from the plate and eluting

with a strong solvent [244].

TLC has the advantage of being a highly cost-effective qualitative technique in as

much as a large number or samples can be analysed or separated simultaneously.

Although it is a very simple and convenient technique, one of its limitations is that it

cannot tell the difference between enantiomers and some isomers. Another

disadvantage of TLC is that in order to identify specific compounds, the Rf values for

the compounds of interest must be known beforehand [245].

236

10.1.4.2 Liquid chromatography (column)

Column chromatography is the most common form of chromatography. Column

chromatography is suitable for the physical separation of microgram to kilogram

quantities of material. A solvent acts as the mobile phase while a finely divided solid

surface acts as the stationary phase. The stationary phase will adsorb the components

of the mixture to varying degrees. As the solution containing the mixture passes over

the adsorbent, the components are distributed between the solvent and adsorbent

surface. This process may be described by a three-way equilibrium between the

sample, the solvent and the adsorbent [48]. The solvent and sample compete for

positions on the solid adsorbent, the solvent displacing the sample reversibly and

continuously in the direction of the solvent flow. Consequently, a weakly adsorbed

compound will spend more time in the solvent, and will therefore be eluted first. The

stationary phase is solid and packed in a column made of glass, stainless steel, or

other inert material. The common solid adsorbents are alumina (aluminum oxide) and

silica gel (silicon dioxide). The sample mixture is applied to the top of the column

and the mobile phase passes through the column either by gravity, vacuum, or

pressure [238].

Liquid-solid column chromatography is an effective separation technique when all

appropriate parameters and equipment are used. This method is especially effective

when the compounds within the mixture are colored, as this gives the scientist the

ability to see the separation of the bands for the components in the sample solution.

Even if the bands are not visible, certain components can be observed by other

visualization methods. One method that may work for some compounds is irradiation

237

with ultraviolet light. This makes it relatively easy to collect samples one after

another. However, if the components within the solution are not visible by any of

these methods, it can be difficult to determine the efficacy of the separation that was

performed. In this case, separate collections from the column are taken at specified

time intervals. Since the human eye is the primary detector for this procedure, it is

most effective when the bands of the distinct compounds are visible [246].

10.1.4.3 Size Exclusion Chromatography

Size exclusion chromatography separates molecules by their size. This is done by

having the stationary phase be packed with small particles of silica or polymer to form

uniform pores. The smaller molecules will get trapped in the silica particles and will

elude from the column at a rate that is greater than that of larger molecules. Thus, the

retention time depends on the size of the molecules. Larger molecules will be swept

away in the mobile phase, therefore having a smaller retention time [245]. In this type

RI FKURPDWRJUDSK\ WKHUH LVQ¶W DQ\ LQWHUDFWLRQ EHLQJ SK\VLFDO RU FKHPLFDO EHWZHHQ

the analyte and the stationary phase. The most commonly used size inclusion sorbents

are the dextran gels, particularly the lipophilic versions such as Sephadex LH-20,

which are mostly used for the separation of small hydrophobic natural products from

WKHLUODUJHUµµFRQWDPLQDQWV¶¶XVXDOO\ chlorophylls, fatty acids, and glycerides [48]. In

organic solvents such as chloroform and methanol, these gels swell to form a matrix.

As compounds migrate with the solvent through the gel, small molecules become

included into the gel matrix, whereas larger ones are excluded and migrate at a greater

rate [238].

238

One of the most extensively used gels in natural product separation, especially

nonpolar or intermediate polarity compounds, is Sephadex LH-20, a

hydroxypropylated form of Sephadex G-25. The derivatization offers lipophilicity to

the gel, at the same time preserving its hydrophilicity. As a result of the added

lipophilicity, LH-20 gel swells sufficiently in organic solvents and allows handling of

natural products that are soluble in organic solvents [245]. The useful fractionation

range of LH-20 is approx. 100±4000 amu, and is particularly ideal for removal of

chlorophyll from plant extracts. It should be noted that separations on gels such as

Sephadex LH-20 also involve the mechanisms of adsorption, partition, possibly ion

exchange, and occasionally the trend of larger molecules eluting first and smaller ones

eluting last may be reversed [245]. The gel filtration mode is operational when a

single eluent is used. The partition mechanism comes into play when the eluent is a

mixture of solvents (usually a mixture of polar and nonpolar solvents); the more polar

of the solvents is taken up by the gel, resulting in a two-phase system with stationary

and mobile phases of different compositions [238] .

This form of chromatography has found considerable use in the removal of

µLQWHUIHULQJ¶SODQWSLJPHQWVVXFKDVWKHFKORURSK\OOV that tend to be larger and more

lipophilic than many plant natural products. Sephadex LH-20 has also been used for

the separation of various avermectins and is the most common method for the

preparative isolation of condensed and hydrolysable tannins [247]. This is a non-

destructive method for the recovery of a high quantity or extract [48].

239

10.1.4.4 High performance liquid chromatography system

HPLC instrumentation includes a pump, injector, column, detector and recorder or

data system. The heart of the system is the column where separation occurs. Since the

stationary phase is composed of micrometer size porous particles, a high pressure

pump is required to move the mobile phase through the column. The components (or

analytes) are first dissolved in a solvent, and then forced to flow through a

chromatographic column under a high pressure. The chromatographic process begins

by injecting the solute onto the top of the column where the mixture is resolved into

its components [243]. The amount of resolution is important and is dependant upon

the extent of the interaction between the solute components and the stationary phase.

The stationary phase is defined as the immobile packing material in the column.

Separation of components occurs as the analytes and mobile phase are pumped

through the column. Eventually each component elutes from the column as a narrow

band (or peak) on the recorder [245]. The interaction of the solute with the mobile and

stationary phases can be manipulated through different choices of both solvents and

stationary phases. As a result, HPLC acquires a high degree of versatility not found in

other chromatographic systems and it has the ability to easily separate a wide variety

of chemical mixtures [243].

Modern HPLC uses a non-polar solid phase, like C-18, and a polar liquid phase,

generally a mixture of water and another solvent. High pressures up to 400 bars are

required to elute the analytes through the column before they pass through a diode-

array detector. The detector measures the absorption spectra of the analytes to aid in

their identification. HPLC is useful for compounds that cannot be vaporized or that

240

decompose under high temperature [243]. Initially, pressure was selected as the

principle criterion of modern liquid FKURPDWRJUDSK\ DQG WKXV WKH QDPH ZDV µKLJK

SUHVVXUHOLTXLGFKURPDWRJUDSK\¶7KLVZDV however, an unfortunate term because it

seems to indicate that the improved performance is primarily due to the high pressure.

This is not true. In fact, high performance is the result of many factors: very small

particles of narrow distribution range and uniform pore size and distribution, high

pressure column slurry packing techniques, accurate low volume sample injectors,

sensitive low volume detectors and good pumping systems [243].

There is a growing interest in the chemical composition of plants and the use of

HPLC has several advantages over other methods. The technique is sensitive, rapid

and does not require the preparation of derivatives before separation. A further

advantage is that ultraviolet absorption detectors set in the aromatic region of the

spectrum (e.g. 275 nm) may also be used. Several reports have also been published of

the application of HPLC to various phenolic acids and aldehydes that occur in plants

[248].

10.1.5 Structure elucidation

In most cases of extraction and isolation of natural products, the end point is the

identification of the compound or the conclusive structure elucidation of the isolated

compound. However, structure elucidation of compounds isolated from plants, fungi,

bacteria, or other organisms is generally time consuming, and sometimes can be the

µµERWWOHQHFN¶¶ LQQDWXUDO SURGXFW UHVHDUFK [48]. There are many useful spectroscopic

methods of getting information about chemical structures, but the interpretation of

241

these spectra normally requires specialists with detailed spectroscopic knowledge and

wide experience in natural product chemistry. With the remarkable advances made in

the area of artificial intelligence and computing, there are a number of excellent

automated structure elucidation programs available that could be extremely useful

[238].

If the target compound is known, it is often easy to compare preliminary

spectroscopic data with literature data or to make direct comparison with the standard

sample. However, if the target compound is an unknown and complex natural

product, a comprehensive and systematic approach involving a variety of physical,

chemical, and spectroscopic techniques is required [48]. Information on the chemistry

of the genus or the family of plant or microbe under investigation could sometimes

provide additional hints regarding the possible chemical class of the unknown

compound [249]. Some of the spectroscopic methods used for structure elucidation

include ultraviolet-visible spectroscopy (UV-vis), infrared spectroscopy (IR), mass

spectrometry (MS) and nuclear magnetic resonance (NMR). In this study we have

used NMR.

10.1.5.1 Nuclear magnetic resonance (NMR)

Nuclear magnetic resonance (NMR) is a spectroscopic technique involving a

magnetic field in which a sample is placed. The sample is then subjected to

radiofrequency radiation at the appropriate frequency, allowing for the absorption of

energy depending on the type of nucleus, whether, for example, it is a 1H or 13C. The

frequency also depends on chemical environment of the nucleus, whether methyl or

242

hydroxyl protons are present, molecular conformations, and dynamic processes. Both

1H NMR and 13C NMR are used, with the latter providing information on the carbon

skeleton, and the former relating to the specifics of the hydrogen atoms, thus

complementing each other [245].


10.2.1 Isolation of compound 1-5 (crude fractionation)

The isolation scheme for compound 1-5 is shown in Figure 10.1. The ethanol extract

of the leaves (LE-40 g) was dissolved in water. It is then partitioned with petroleum

ether, chloroform, and water to yield the respective solvent extracts (LE-PE, LE-CL

and LE-WA). Phytochemical analysis was repeated as in 3.2 (iv) to confirm the

absence of tannins in LE-PE and LE-CL and the presence of tannins in LE-WA. The

solvent partitioning thus concentrates all tannins in the water fraction.

10.2.2 Isolation of compound 1 & 2

10.2.2.1 Silica gel column chromatography

A glass column was clamped upright and packed with silica gel (size 0.063-0.200

mesh) mixed with the appropriate mobile phase and poured into the column (4 cm x

90 cm) as a compact even suspension. This constituted the stationary phase. The

extract (LE-CL weighing 6 g) was then mixed with a small amount of chloroform

243

and introduced as a thin band to the silica gel. Once the extract was loaded onto the

silica gel, the mobile phase was added at a constant flow rate. Gradient elution of

increasing polarity was initiated with chloroform/methanol gradient elution (the ratio

from 100:0 to 92:8). Thirteen column fractions were collected and analyzed by TLC

(chloroform/methanol). Fractions with similar TLC pattern were combined to total of

four fractions.

10.2.2.2 Preparative thin layer chromatography (PTLC)

Fraction 2 that was yielded from chloroform/ methanol ratio 96:4 was

rechromatographed on a preparative TLC (silica gel plate with 2 mm thickness) with

solvent system chloroform/ methanol (ratio of 1000:15) yielding total 7 bands. Band

five was collected and rechromatographed on preparative TLC (0.5 mm thickness)

with solvent system chloroform/ methanol (ratio of 89:11) to yield four bands, with

band two and band three sent for NMR characterisation.

10.2.3 Isolation of compound 3, 4 & 5

10.2.3.1 Size-exclusion column chromatography

The principle governing this method is based on the molecular size differences of the

compounds, which result in the separation of these compounds. The stationary phase

comprised of a porous three-dimensional polymeric matrix, namely SephadexTM LH-

20 (GE Healthcare). This matrix was initially saturated with ethanol in order to

facilitate swelling before use. SephadexTM LH-20 in ethanol adsorbs tannins while it

244

desorbs tannins in acetone [247]. This is then useful for the separation of tannin and

non-tannin containing fractions. Once swelling was achieved the matrix was then

introduced into a glass column and the fraction (LE-WA) which was dissolved in

ethanol was introduced as a thin band was then applied to the column. The mobile

phase consisted ethanol. Sixteen fractions were collected analyzed by TLC. Fractions

with similar TLC pattern were combined to total of four fractions. The adsorbed

tannins were then flushed with a change of mobile phase to acetone:water (70:30)

resulting in fraction WT. The phytochemical tannin test (section 3.2-iv) was again

performed to confirm the presence of tannins in WT and absence in the other

fractions. The matrix had irreversibly sorbed material that could be tannin [247].

Fraction WB was reintroduced to another column with SephadexTM LH-20 that was

swelled in methanol. Total 3 fractions were collected and fraction WB2 was sent for

nuclear magnetic resonance (NMR) characterisation. Fraction WD was also

introduced to SephadexTM LH-20 that was swelled in methanol. Total five fractions

were collected and fraction WD3 and WD4 were sent for nuclear magnetic resonance

(NMR) characterisation.

10.2.4 Isolation of compound 6-8

10.2.4.1 Isolation of compound 6-8 (PTLC and solvent partioning)

The isolation scheme for compound 6-8 is shown in Figure 10.2. The chloroform

extract of the barks were dissolved in dicloromethane:methanol (2:1). Mixture was

chromatographed on a PTLC (silica gel plate with 2 mm thickness) with solvent

245

system ethyl acetate: methanol (10:1) yielding total three bands (Figure 10.3). First

band (B1) was rechromatographed on PTLC (2 mm thickness) with solvent system

ethyl acetate: methanol (6:1) yielding four bands. The third band upon reconstitution

resulting in fraction B1-BA was subjected to HPLC (section 10.4.5). The second band

(B2) from Figure 10.3 was rechromatographed on PTLC (2 mm thickness) with

solvent system ethyl acetate: methanol (6:1) yielding three bands. The second band of

this PTLC plate upon reconstitution (B2-BA) was subjected to HPLC (section 10.4.5).

The third band (B3) from Fig 10.3 and all the other areas including origin was

combined and partitioned between water and ethyl acetate resulting in aqueous (B3-

AL) and organic layer (B3-OL). The B3-AL fraction was subjected to HPLC (section

10.4.5).

10.2.4.2 Isolation of compound 6-8 (HPLC)

Fraction B1-BA, B2-BA and B3-AL was concentrated in vacuum and then dissolved

in methanol. The solution was then subjected to HPLC individually. The samples

were then qualitatively analyzed by using Agilent 2690 semi-prep HPLC system

(Luna C-18 column, 100 mm x 30 mm) equipped with a 996 photodiode-array

detector (PDA). The injection of the sample was 5 µl and the flow rate was 2 ml/min.

246

Liquid- liquid partitioning

With petroleum ether with chloroform with water

Silica gel 60 SephadexTM LH-20 Final flushing Chloroform:methanol column with (100:0 92:8) Ethanol acetone:water

13 fractions recombined 16 fractions recombined (70:30) to 4 fractions to 4 fractions

PTLC SephadexTM-LH 20 Chloroform:methanol Methanol (1000:15) 7 fractions

PTLC Chloroform:methanol (89:11)

Figure 10.1: Schematic representation of the isolation and purification of compound 1 (scopoletin), compound 2 (scoparone), compound 3 (+-catechin), compound 4 (hyperin) and

compound 5 (cynaroside) isolated from Canarium patentinervium Miq.

Ethanol extract of leaves, LE Dissolved in water ± 40 g

Extraction of Canarium patentinervium Miq. bark and leaves (500 g)

Crude hexane extract Crude chloroform extract Crude ethanol extract

LE-PE ± 11 g LE-CL ± 6 g LE-WA ± 24 g

2% met

CA

4% met

CB

6% met

CC

8% met

CD

CB1 CB2 CB3 CB4 CB5 CB6 CB7

CB4-1 CB4-2 CB4-3 CB4-4

Compound 1 (scopoletin)

49 mg

Compound 2

(scoparone)

11 mg

WA WB WC WD

Final

fraction

WT

WB1 WB2 WB3

Compound 3

(+-catechin)

14 mg

WD1 WD2 WD3 WD4 WD5

Compound 4

(hyperin)

5.2 mg

Compound 5

(cynaroside)

4.8 mg

247

PTLC Ethyl acetate:methanol (10:1)

PTLC PTLC Combined and partitioned Ethyl acetate: methanol Ethyl acetate:methanol between water and (6:1) (6:1) ethyl acetate

Concentrated in vacuum Concentrated in vacuum Concentrated in vacuum Dissolved in methanol Dissolved in methanol Dissolved in methanol 5 ml of solution 5 ml of solution 5 ml of solution Semi-Prep HPLC Semi-Prep HPLC Semi-Prep HPLC Mobile phase Mobile phase Mobile phase (A: water, B: acetonitrile) (A:water, B:acetonitrile) (A:ammonium bicarbonate, B: acetonitrile)

Acetonitrile removed by Acetonitrile removed by Acetonitrile removed by Evaporation and evaporation and evaporation and freeze dried freeze dried freeze dried

Dissolved in methanol Dissolved in methanol Dried under nitrogen Dried under nitrogen

Figure 10.2: Schematic representation of the isolation and purification of compound 6 (vomifoliol), compound 7 (lioxin) and compound 8 syringic acid isolated from Canarium

patentinervium Miq.

Chloroform extract of barks (BC) Dissolved in dicloromethane:methanol

(2:1) -7.2g

Extraction of Canarium patentinervium Miq. bark and leaves (500 g)

Crude hexane extract Crude chloroform extract Crude ethanol extract

Band 1, B1 Band 2, B2 Band 3, B3 Others, BO

B1-BA Others

Fraction

(Rt= 14.1

min)

B1-14 (Solid)

Compound 6

(vomifoliol) -15 mg

B2-BA

Fraction

(Rt= 15.8

min)

B2-15 (Solid)

Compound 7

(lioxin) ± 3.4 mg

Others Aqueous layer

(B3-AL)

Organic

layer

Fraction (Rt=

3.3 min)

B3-3 (Solid)

Compound 8 (syringic

acid) ± 3.6 mg

248

Figure 10.3: The PTLC of chloroform extract of barks

Solution B1-BA: The mobile phase started off with 15 % acetonitrile and 85 % water.

The solvent ratio was changed through a linear gradient to 20 % acetonitrile and 80 %

water in 14 min. This ratio was maintained for 2 min and thereafter the solvent ratio

was changed back to the initial starting conditions. At Rt (retention time) = 14.1 min

the fraction eluted (B1-14) was collected and the acetonitrile was removed by

evaporation and freeze-drying. The solid in the vial was then redissolved in methanol

and dried under nitrogen. B1-14 was then sent for nuclear magnetic resonance (NMR)

characterisation.

Solution B2-BA: The mobile phase started off with 15 % acetonitrile and 85 % water.

The solvent ratio was changed through a linear gradient to 20 % acetonitrile and 80 %

water in 14 min. This ratio was maintained for 4 min and thereafter the solvent ratio

was changed back to the initial starting conditions. At Rt (retention time) = 15.8 min

the fraction eluted (B2-15) was collected and the acetonitrile was removed by


Diagram of TLC

249


characterisation.

Solution B3-AL: The mobile phase started off with 5 % acetonitrile and 95 % water

(containing 10 mM ammonium bicarbonate). The solvent ratio was changed through a

linear gradient to 16% acetonitrile and 84 % water (containing 10 mM ammonium

bicarbonate) at 1 min. This ratio was maintained for 5 min and thereafter the solvent

ratio was changed back to the initial starting conditions. At Rt (retention time) = 3.3

min the fraction eluted (B3-3) was collected and the acetonitrile was removed by



characterisation.

10.2.5 Nuclear magnetic resonance (NMR)

Final chemical characterisation of the 8 isolated compounds was achieved by NMR,

which was performed in collaboration with Assistant Professor Dr. Achyut Adhikari

(HEJ Research Institute of Chemistry, University of Karachi, Pakistan). Nuclear

magnetic resonance spectroscopy was performed on a Varian 500 MHz NMR

spectrometer. Samples CB4-2 and CB4-3 were recorded at room temperature in

deuterated chloroform (CDCL3), samples B1-14,B2-15, WB-2, WD-3 and WD-4

were recorded at room temperature in deuteromethanol (CD3OD) while sample B3-3

were recorded at room temperature in deuterated water (D2O).

250

10.3 Results

Compound 1 was isolated as a pale yellow powder. On the basis of the spectral data

obtained for 1H NMR and 13C NMR [Table 10.1 and Figure A1 & A2 (Appendix A )],

and compared to that of relative references [250][251][252], compound 1 was

identified as scopoletin (Figure 10.4). It has an empirical formula of C10H8O4 with

molecular weight of 192.16.

Table 10.1: 1H and 13C NMR spectral data (CDCL3, 500 MHz) of scopoletin

Position įC įH H's Type J (Hz)

2 161.6 - - - -

3 111.6 6.3 1H d 9.5

4 143.3 7.63 1H d 9.5

5 103.2 6.87 1H s -

6 144 - - - -

7 150.2 - - - -

8 107.4 6.95 1H s -

9 149.7 - - - -

10 113.5 - - - -

6- OCH3 56.4 3.98 3H s -

Figure 10.4: The chemical structure of compound 1 (scopoletin)

251


obtained for 1H NMR and 13C NMR [Table 10.2 and Figure A3 & A4 (Appendix A)],

and compared to that of relative references [253][254], compound 2 was identified as

scoparone (Figure 10.5). It has an empirical formula of C11H10O4 with molecular

weight of 206.19.

Table 10.2: 1H and 13C NMR spectral data (CDCL3, 500 MHz) of scoparone


2 161.41 - - - -

3 111.45 6.32 1H d 9.6

4 150.06 7.64 1H d 9.6

5 100.05 6.87 1H s

6 152.87 - - - -

7 146.37 - - - -

8 143.28 6.88 1H s

9 113.59 - - - -

10 107.98 - - - -

6-OCH3 56.38 3.95 3H s -

7-OCH3 56.4 3.98 3H s -

Figure 10.5: The chemical structure of compound 2 (scoparone)

252

Compound 3 was isolated as light yellow needles. On the basis of the spectral data



catechin. Catechin exists in nature as (+) and (-) enantiomers. The optical rotation of

the compound in methanol was tested using Schmidt + Haensch Polartronic H532

Polarimeter. The experimental rotation was an average of +69.25 (n=4) concluding

that this compound is (+)-catechin (Figure 10.6). Previous published data on (+)-

catechin reports an optical rotation of +56.60 [257]. It has an empirical formula of

C15H14O6 with molecular weight of 290.26.

Table 10.3: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of (+)-catechin


2 81.46 4.01 1H d 7.5

3 67.41 3.98 1H m 5.5, 8.5

4 27.12

4a - 2.52 1H dd 8.2, 16.1

4b - 2.87 1H dd 5.5, 16.1

5 156.19 - - - -

6 94.86 5.94 1H d 2.3

7 156.45 - - - -

8 94.08 5.87 1H d 2.3

9 155.52 - - - -

10 99.40 2.52 1H dd -

OH - 4.58 1H d -

1' 130.82 - - - -

2' 113.84 6.85 1H d 1.8

3' 144.83 - - - -

4' 144.85 - - - -

5' 114.66 6.78 1H d 8.1

6' 118.62 6.74 1H dd 1.8, 8.1

253

Figure 10.6: The chemical structure of compound 3 [(+)-catechin]




hyperin (Figure 10.7). It has an empirical formula of C21H20O12 with molecular

weight of 460.37.

Figure 10.7: The chemical structure of compound 4 (hyperin)

254

Table 10.4: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of hyperin


2 157.37 - - - -

3 134.36 - - - -

4 178.13 - - - -

5 161.63 - - - -

6 98.55 6.23 1H d 2.10

7 164.90 - - - -

8 93.34 6.43 1H d 2.10

9 157.08 - - - -

10 104.17 - - - -

1' 121.51 - - - -

2' 114.67 7.86 1H d 2.60

3' 144.43 - - - -

4' 148.56 - - - -

5' 116.36 6.88 1H d 8.60

6' 121.51 7.60 1H dd 2.6, 8.6

1'' 103.99 5.19 1H d 8.20

2'' 71.77 3.84 1H dd 8.2, 8.0

3'' 75.80 3.60 1H dd 8.0, 2.1

4'' 68.62 3.87 1H dd 3.2, 2.1

5'' 77.88 3.45 1H m -

6'' 60.53 - - - -

6a'' - 3.67 1H dd 11.0, 3.0

6b'' - 3.56 1H dd 11.0, 5.8

Compound 5 was isolated as a light yellow powder. On the basis of the spectral data

obtained for 1H NMR and 13C NMR [Table 10.5 and Figure A9 & A10 (Appendix

A)], and compared to that of relative references [260][261], compound 5 was

identified as cynaroside (Figure 10.8). It has an empirical formula of C21H20O11 with

molecular weight of 448.38.

255

Table 10.5: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of cynaroside


2 165.52 - - - -

3 102.72 6.60 1H s

4 182.66 - - - -

5 161.51 - - - -

6 100.27 6.53 1H d 2.00

7 163.43 - - - -

8 94.65 6.83 1H d 2.00

9 157.58 - - - -

10 105.69 - - - -

1' 121.98 - - - -

2' 112.81 7.43 1H d 2.20

3' 145.77 - - - -

4' 149.80 - - - -

5' 115.40 6.93 1H d 8.40

6' 119.12 7.45 1H dd 8.40, 2.20

1'' 99.75 5.09 1H d 7.30

2'' 73.33 3.48 1H t 9.20

3'' 76.46 3.56 1H t 9.20

4'' 69.86 3.45 1H t 9.20

5'' 77.00 3.60 1H m -

6'' 61.06 - - - -

6a'' - 3.75 1H dd 12.40, 5.80

6b'' - 3.95 1H dd 12.40, 1.90

Figure 10.8: The chemical structure of compound 5 (cynaroside)

256

Compound 6 was isolated as white solid (HPLC chromatogram in Figure B1-

Appendix B). On the basis of the spectral data obtained for 1H NMR and 13C NMR

[Table 10.6 and Figure A11 & A12 (Appendix A)], and compared to that of relative

references [262][263], compound 5 was identified as vomifoliol (Figure 10.9). It has

an empirical formula of C13H20O3 with molecular weight of 224.30.

Table 10.6: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of vomifoliol


1 78.54 - - - -

2 41.03 - - - -

3a - 2.18 1H d 15.00

3b - 2.54 1H d 15.00

4 199.85 - - - -

5 125.69 5.90 1H m -

6 166.10 - - - -

7 128.71 5.81 1H m -

8 135.49 5.82 1H m -

9 67.34 4.34 1H m -

10 22.41 1.26 3H d 7.50

11 23.06 1.03 3H s -

12 22.05 1.06 3H s -

13 18.15 1.94 3H s -

Figure 10.9: The chemical structure of compound 6 (vomifoliol)

257

Compound 7 was isolated as white powder (HPLC chromatogram in Figure B2-



references [264][265], compound 7 was identified as lioxin (Figure 10.10). It has an

empirical formula of C8H8O3 with molecular weight of 152.15.

Table 10.7: 1H and 13C NMR spectral data (CD3OD, 500 MHz) of lioxin

Position įC įH H's Type

1 127.72 - - -

2 110.07 7.30 1H m

3 149.09 - - -

4 147.08 - - -

5 115.51 7.30 1H d

6 125.64 7.31 1H m

7 191.60 9.57 1H s

OH - 6.77 1H s

10 54.80 3.80 3H s

Figure 10.10: The chemical structure of compound 7 (lioxin)

258

Compound 8 was isolated as pale yellow powder (HPLC chromatogram in Figure B3-



references [266][267], compound 8 was identified as syringic acid (Figure 10.11). It

has an empirical formula of C9H10O5 with molecular weight of 198.17.

Table 10.8: 1H and 13C NMR spectral data (D2O, 500 MHz) of syringic acid

Position įC įH H's Type

1 121.08 - - -

2 106.92 7.11 1H s

3 147.08 - - -

4 148.41 - - -

5 147.08 - - -

6 106.92 7.11 1H s

2-OCH3, 6-OCH3 56.26 3.57 3H s

COOH 174.29 - - -

Figure 10.11: The chemical structure of compound 8 (syringic acid)

259

10.4 Discussion

All the compounds were isolated for the first time from Canarium patentinervium

Miq. Seven phenolic compounds (compounds 1-6 and 8) and one norsesquiterpene

with cyclohexenone ring (compound 7) was isolated.

The 1H-NMR spectrum of compound 1 (Table 10.1) revealed the presence of two

doublets at įH 7.63 (H-4, J = 9.5 Hz) and 6.30 (H-3, J = 9.5 Hz). The two singlet

signals appeared at įH 6.87 and 6.95 were assigned for the two protons H-5 and H-8,

respectively. The 1H NMR spectra of compound 2 identified as scoparone (Table

10.2) showed two doublets with coupling constant of 9.+]DWįDQGį 7.64,

which were assigned as H-3 and H-4, respectively, characteristic for coumarins [253].

The 1H NMR spectrum of scoparone was similar to that of scopoletin, except for an

additional siQJOHWDWįppm indicating its identity as scoparone [268].

Compound 3 were isolated as light yellow crystals. The structural identity of this

compound was determined through NMR spectral analysis (Table 10.3) that was

compared to literature [255][256]. Catechin possesses two benzene rings (called the

A- and B-rings) and a dihydropyran heterocycle (the C-ring) with a hydroxyl group

on carbon 3. The A ring is similar to a resorcinol moiety while the B ring is similar to

a catechol moiety. There are two chiral centers on the molecule on carbons 2 and 3

resulting in 4 optical isomers (+) catechin, (-) catechin, (+) epicatechin and (-)

epicatechin. Of these, (+) catechin and (-) epicatechin occur in nature [269]. From the

experimental data of optical rotation, this compound was confirmed to be (+)

catechin.

260

Compound 4 was isolated as pale yellow powders. In the 1H-NMR spectrum, the

typical proton signals of quercetin moiety, that is, those of an AMX system due to a

1',3',4'-WULVXEVWLWXWLRQRIULQJ%>į 7.86 (d, J = 2.+]GG- +]DQGį

6.88 (d, J = 8.6 Hz)], and a typical meta-coupled pattern for H-8 and H-SURWRQV>į

6.43 (d, J = 2.1 Hz) and 6.23 (d,J = 2.1 Hz)], were observed. The 13C-NMR of

compound 4 showed resonances for twenty one carbons and presence of a quercetin

moiety with the exception of monosaccharide moiety (Table 10.4). An anomeric

proton signals of compound 4 DSSHDUHGDWį 5.19 (d, J = 8.2 Hz) and other oxygenated

methines and methylene signals RIVXJDUPRLHW\DWį (dd, J = 3.2, 2.1 Hz, H-4''),

3.84 (dd, J = 8.2, 8.0 Hz, H-2''), 3.67 (dd, J = 11.0, 3.0, H-6''a), 3.56 (dd, J = 11.0, 5.8

Hz, H-6''b), 3.60 (dd, J = 8.0, 2.1 Hz, H-3'') and 3.45 (m, H-5''). Also, the carbon

VLJQDOVRIWKHVXJDUPRLHW\ZHUHREVHUYHGDWį 103.99 (C-1''), 77.88 (C-5''), 75.80 (C-

3''), 71.77 (C-2''), 68.62 (C-4'') and 60.53 (C- VXJJHVWLQJ WKH SUHVHQFH RI ȕ-

galactopyranoside units. Thus, the structure of compound 4 was identified as

quercetin-3-O-ȕ-D-galactopyranoside, hyperin [258].

The 1H NMR spectrum of compound 5, a light yellow powder, showed two meta±

coupled doublets (J=2.0 Hz) at į6.83 and 6.53, each integrating for one proton, and

were assigned to H-8 and H-6, respectively of ring A of 5,7-dihydroxyflavonoids

[270]. The presence of ABX system at į7.45 (dd, J = 8.4, 2.2 Hz, H-6'), 7.43 (d, J =

2.2 Hz, H-2') and 6.93 (d, J = 8.3 Hz, H-5'), characteristic of 1, 2, 4-trisubstituted

phenyl unit [270]. The only singlet at į6.60, integrating for one proton, was attributed

C-3 to proton of flavonoids (35). These spectral data revealed the presence of luteolin

skeleton [271]. In addition, the 1H NMR spectrum showed a series of signals between

261

įGG- +]+-6''b), 3.75 (dd, J = 12.40, 1.90 Hz, H-6''a), 3.60 (t,

H-5''), 3.56 (t, J = 9.2 Hz, H-3''), 3.48 (dd, J = 9.2, H-2'') and 3.45 (t, J = 9.2 Hz, H-4'')

attributable to a sugar moiety. The coupling constant (J = 7.3 Hz) of the anomeric

proton ORFDWHG DW į (d, J = 7.3 Hz, H-1'') and the 13C NMR (Figure 10.8)

chemical shifts of the sugar carbons į and 61.06)

UHYHDOHGWKHSUHVHQFHRIȕ-O-glucoside unit in luteolin- 7-O-glucoside. The 13C NMR

data showed the presence of a ketone carbonyl at C-į), two olefinic carbons

at C-2 and C-3 įDQG), and four hydroxyl carbons at C-7, C-5, C-4'

and C-į, 161.51, 149.80 and 145.77). On comparison with the literature on

flavonoids of luteolin, it was revealed that the physical and spectral data of luteolin-7-

O-glucoside isolated were in good agreement with those recorded for luteolin- 7-O-

glucoside or cynaroside [271][260].

The comparison of the corresponding NMR data of compound 6, a white solid (Table

10.6) with those reported in literature [262][263] led to its identification as vomifoliol.

The cyclohexenone structure was confirmed by the observation in the HMBC (Figure

A13-Appendix A) spectrum of correlations between the AB system H-3ab (įį

2.54, J = 15.00 Hz) and C-4 į ), C-1 į ) and C-5 į .69). The

location of the two high field methyl groups C-11 and C-12 at C-2 was ascertained by

the detection of the H3ab-C11 and H3ab-C12 correlations. The position of the side

chain on C-1 of the cyclohexenone ring is confirmed by the observation in the HMBC

spectrum of the H7-C1 correlation. The COSY (Figure A14-Appendix A) spectrum

revealing FRUUHODWLRQVEHWZHHQWKHROHILQLFVLJQDO+įZLWKWKHRWKHUROHILQLF

proton H8 į) which in turn had a cross peak with H9 į) permitted us to

262

confirm the structure of this side chain. All these NMR data together suggested the

structure of vomifoliol.

The 1H NMR spectrum of compound 7 which was isolated as white powder exhibited

the following signals (Table 10.7)į (CHO), multiplets at į 7.30, 7.31 (2H) and

į GRXEOHW DW į 7.04 (1H) (aromatic protons), and 3.80 ppm (CH3O). The 13C NMR

VSHFWUXPVKRZHGVLJQDOVDWį (CHO), 149.09, 147.08, 127.72, 125.64, 115.51,

and 110.07 (aromatic carbons) and 54.80 ppm (CH3O). The 1H and 13C spectra were

identical to those of 4-hydroxy-3-methoxybenzaldehyde (Figure 10.10) (also known

as vanillin or lioxin) [265].

Compound 8 eluted first to give a pale yellow powder on drying. The 13C NMR

showed two methoxyls, two olefinic methines and five quaternary carbons, including

a carboxy, and three oxygenated olefinic carbons. The only signals appearing in the

1H NMR were a 6H singlet for the two methoxyls (į) and a 2H singlet for two

aromatic protons (į 7.11) (Table 10.8). Further analysis of the NMR data indicated a

symmetric trisubstituted phenolic acid. Two of the substituents were methoxyl groups

and the third a hydroxyl group. The data was found to fit well with that of 4-hydroxy-

3,5-dimethoxybenzoic acid (syringic acid) [266][267]. Further evidence for the

structure of 8 was provided by a 3-bond correlation between H-2/6 (į7.11) and the

carboxy carbon (į 174.29) (Figure 10.11). The possibility of 4-hydroxy-2,6-

dimethoxybenzoic acid was thus eliminated.

263

10.5 Conclusion

Eight compounds were isolated for the first time from Canarium patentinervium Miq.

using various isolation techniques such as TLC, CC, HPLC and identified with NMR

method. Compounds isolated scopoletin, scoparone, (+)-catechin, hyperin,

cynaroside, lioxin and syringic acid were phenolics while vomifoliol was a

norsesquiterpene with a cyclohexenone ring. Lioxin, syringic acid and vomifoliol

were isolated from this genus for the first time. These findings agree with the

phytochemical analysis which was done in Chapter 2.

264

CHAPTER 11

BIOACTIVITY OF ISOLATED SECONDARY METABOLITES

11.1 Introduction

11.1.1 Terpenes and phenols

Terpenes are the most numerous and structurally diverse plant natural products. The

term terpene usually refers to a hydrocarbon molecule while terpenoid refers to a

terpene that has been modified, such as by the addition of oxygen. Terpenoids

(sometimes called as isoprenoids) are hydrocarbon natural products based on 5-

carbon (isoprene) units as their building blocks [237]. Terpenes are an enormous class

of natural products spanning well over 30,000 members having been discovered to-

date [272]. These compounds are classified as to the number of isoprene units, which

include the hemiterpenes (1), monoterpenes (2), sesquiterpenes (3), diterpenes (4),

sesterterpenes (5), tripterpenes (6), and polyterpenes (many units). Sometimes the

prefix nor- is added to name a structural analog that can be derived from a parent

compound by the removal of one carbon atom along with the accompanying hydrogen

atoms (norsesquiterpene) [237].

Of these different major classes of terpenoids, the diterpenes are structurally most

diversified, possessing at least 6 large structural groups. Biological active compounds

are found in each class of the terpenoids, particularly among the sesquiterpenes,

diterpenes and triterpenes [237]. Isopentyl diphosphate (IPP) and dimethyl allyl

265

diphosphate (DMAPP) are found to be the actual building blocks in terpene

biosynthesis [273]. There are 2 biosynthetic pathways for the production of IPP and

DMAPP, the mevalonate pathway and the more recently discovered deoxyxylulose

pathway. All terpenes are formed through the reactions of IPP and DMAPP [237].

Rare terpenoids such as norisoprenoids, like the C13-norisoprenoids 3-oxo-Į-ionol are

found to be present in Muscat of Alexandria leaves and 7,8-dihydroionone

derivatives, and megastigmane-3,9-diol and 3-oxo-7,8-dihydro-Į-ionol found in

Shiraz leaves (both grapes in the species Vitis vinifera) or wine (responsible for some

of the spice notes in Chardonnay) [274].

Polyphenols are the third most widespread class of metabolites in nature, and their

distribution is almost ubiquitous [241]. It is estimated that 100,000 to 200,000

secondary metabolites exist and some 20% of the carbon fixed by photosynthesis is

channeled into the phenylpropanoid pathway, thus generating the majority of the

natural-occurring phenolics, such as flavonoids and stilbenes [275].. Although a large

variety of plant phenols exists, most of these compounds arise from a common origin:

the amino acids phenylalanine or tyrosine. These aminoacids are deaminated to

cinnamic acids, which enter the phenylpropanoid pathway. As a general rule recently

proposed by Quideau et al. [276]WKHWHUPµSODQWSKHQROLFV¶VKRXOGEHVWULFWO\XVHGWR

refer to secondary natural metabolites arising biogenetically from the shikimate/

phenylpropanoid pathway, which directly provides phenylpropanoids (Figure 11.1),

RUWKHµSRO\NHWLGH¶acetate/malonate pathway, which can produce simple phenols, or

both of them [277]. A key step in this biosynthetic route is the introduction of one or

more hydroxyl groups into the phenyl ring. As result, these compounds are derived

from a common carbon skeleton building block: the C6-C3 phenylpropanoid unit [53].

266

Biosynthesis, according to this pathway, produces the large variety of plant phenols:

cinnamic acids (C6-C3), benzoic acids (C6- C1), flavonoids (C6-C3-C6),

proanthocyanidins [(C6-C3-C6)n], coumarins (C6-C3), stilbenes (C6-C2-C6), lignans

(C6-C3-C3-C6) and lignins [(C6-C3)n]. Main classes of phenolics include phenolic acid

(benzoic acid derivatives and cinnamic acid derivatives), flavonoids (flavones,

flavonols, flavanones, flavan-3-ols, anthocyanidins and isoflavones), coumarins,

stilbenes and lignans (Figure 11.2) [275]. These pathways produce a bewildering

array of monomeric and polymeric VWUXFWXUHVWKHWHUPµSRO\SKHQROV¶GHILQLQJWKRVH

with more than one phenolic ring) that fulfill a very broad range of physiological roles

in plants [277]. Although the bulk of these compounds play cell wall structural roles,

plant tissues synthesize a vast array of non-structural constituents that have various

roles in plant growth DQGVXUYLYDO7KXVWKHH[SUHVVLRQ³SODQWSKHQROLFV´HPEUDFHVD

highly diverse group with an extremely large structural diversity: tens of thousands of

diverse structures have been identified, with the number continually increasing

[276][278].

11.1.2 General antioxidant mechanisms of phenolics

Phenolics are able to act as antioxidants in a number of ways. Phenolic hydroxyl

groups are good hydrogen donors: hydrogen-donating antioxidants can react with

reactive oxygen and reactive nitrogen species in a termination reaction, which breaks

the cycle of generation of new radicals [279]. Following interaction with the initial

reactive species, a radical form of the antioxidant is produced, having a much greater

chemical stability than the initial radical. The interaction of the hydroxyl groups of

phenolics with the ʌ-electrons of the benzene ring gives the molecules special

267

properties, most notably the ability to generate free radicals where the radical is

stabilized by delocalization. The formation of these relatively long-lived radicals is

able to modify radical-mediated oxidation processes [280]. The antioxidant capacity

of phenolic compounds is also attributed to their ability to chelate metal ions involved

in the production of free radicals [281]. However, phenolics can act as pro-oxidants

by chelating metals in a manner that maintains or increases their catalytic activity or

by reducing metals, thus increasing their ability to form free radicals [282]. Phenolic

structures often have the potential to strongly interact with proteins, due to their

hydrophobic benzenoid rings and hydrogen-bonding potential of the phenolic

hydroxyl groups. This gives phenolics the ability to act as antioxidants also by virtue

of their capacity to inhibit some enzymes involved in radical generation, such as

various cytochrome P450 isoforms, lipoxygenases, cyclooxygenase and xanthine

oxidase [280][283].

268

Figure 11.1: Schematic of the major branch pathways of (poly) phenol biosynthesis. PAL: phenylalanine ammonia-lyase, C4H: cinnamate-4-hydrolase, 4CL: 4-coumaroyl:CoA-ligase, HCT:

hydroxycinnamoyl transferase, C3H: p-coumarate-3-hyroxylase, CHS: chalcone synthase, CHI: chalcone isomerase, ANS: anthocyanidinstnthase, DFR: dihydroflavonol reductase, FS: flavone

synthase, F3H: Flavanone 3-hydroxylase, IFS: isoflavone synthase, ANR: anthocyanidin reductase, LAR: leucoanthocyanidin reductase [277].

269

Figure 11.2: Main classes of phenolics [275]

270

11.1.3 Flavonoids

Approximately 9000 different flavonoids have been reported from plant sources, and

with almost certainty many more are still to be discovered, as they continue to capture

the interests of scientists from numerous disciplines. Based on the 10-carbon skeleton

of flavonoids, they can be substituted by a range of different groups, viz. hydroxyl,

methoxyl, methyl, isoprenyl and benzyl substituents [284]. Flavonoids are

characterized by a phenylbenzopyran chemical structure. The general structure

includes a C15 (C6-C3-C6) skeleton joined to a chroman ring (benzopyran moiety). The

heterocyclic benzopyran ring is known as the C ring, the fused aromatic ring as the A

ring, and the phenyl constituent as the B ring (Figure 11.2). Commonly flavonoids are

further modified by the addition of substituent groups such as methyl groups,

aromatic acyl groups, and/or sugar moieties [284]. Increasingly, flavonoids are

becoming the subject of medical research. Flavonoids play a role in disease

prevention in humans. They have been reported to possess many useful properties,

including anti-inflammatory, oestrogenic, enzyme inhibition, antimicrobial,

antiallergic, vascular and cytotoxic antitumour activity [285], but the antioxidant

activity is, without a doubt, the most studied one attributed to flavonoids. This well

established antioxidant activity of flavonoids is also responsible for other biological

activities in which the prevention of oxidative stress is beneficial [286].

The anti-oxidants activity of the flavonoids which neutralize damaging chemicals in

the body such as free radicals results in reduction of oxidative stress, which are

thought to contribute to a number of diseases, including atherosclerosis, degenerative

brain GLVHDVHVOLNH$O]KHLPHU¶VGLVHDVHDVZHOODVSUHPDWXUHDJHLQJ [52]. Flavonoids

271

have also been found to help the body fight viruses and decrease allergic reactions.

There is abundant evidence to support the role of flavonoids in disease prevention a

high dietary intake of quercetin is related to a reduced risk of heart disease [287].

Rutin has been found to help prevent stomach ulcers by protecting the stomach lining

and it also protects blood vessels. Flavonoids have also been found to reduce the

µVWLFNLQHVV¶ RI EORRG SODWHOHWV DQG thus reduce the formation of blood clots, which

reduce the risks of certain cardiovascular diseases [288]. Flavan-3-ols are thought to

interfere in the pathogenesis of cardiovascular disease via several mechanisms:

antioxidative, antithrombogenic, and antiinflammatory. In particular,

proanthocyanidins and flavan-3-ol monomers aid in lowering plasma cholesterol

levels, inhibit LDL oxidation, and activate endothelial nitric oxide synthase to prevent

platelet adhesion and aggregation that contribute to blood clot formation [289][290].

The anticancer activity of some flavonoid compounds is due to their ability to

scavenge free radicals, thus avoiding the early stages of cancer promotion. Besides

this mechanism, flavonoids have also been reported to act as anticancer agents via

regulation of signal transduction pathways of cell growth and proliferation,

suppression of oncogenes and tumor formation, induction of apoptosis, modulation of

enzyme activity related to detoxification, oxidation and reduction, stimulation of the

immune system and DNA repair, and regulation of hormone metabolism [289]. In the

past few years we have witnessed the establishment of other flavonoid classes as

potent molecules for the treatment of other pathologies that do not involve these

FRPSRXQGV¶ DQWLR[LGDQW properties. This is the case of some isoflavones, whose

estrogen-like capacity is now well established. The activity of these compounds is

related with their similarity to estradiol estrogen. Genistein and daidzein have

272

demonstrated to be promising molecules for the treatment of conditions in which the

agonist effect in estrogen receptors is beneficial, such as menopause conditions. In

fact, several preparations containing these compounds, mainly soya-derived, are now

used in therapeutics [291].

11.1.4 Phenolic acids

Phenolic acids have a carboxyl group attached or linked to benzene ring (Figure 11.2).

Two classes of phenolic acids can be distinguished depending on their structure:

benzoic acid derivatives (i.e. hydroxybenzoic acids, C6-C1) and cinnamic acid

derivatives (i.e. hydroxycinnamic acids, C6-C3) (Figure 11.2) [292]. Hydroxycinnamic

acid compounds (p-coumaric, caffeic acid, ferulic acid) occur most frequently as

simple esters with hydroxy carboxylic acids or glucose, while the hydroxybenzoic

acid compounds (p-hydroxybenzoic, gallic acid, ellagic acid) are present mainly in the

form of glucosides. Furthermore, phenolic acids may occur in food plants as esters or

glycosides conjugated with other natural compounds such as flavonoids, alcohols,

hydroxyfatty acids, sterols, and glucosides [293].

They have been the subject of a great number of chemical, biological, agricultural,

and medical studies. Among the wide diversity of naturally occurring phenolic acids,

at least 30 hydroxy- and polyhydroxybenzoic acids have been reported in the last 10

years to have biological activities [294]. They have antioxidant, antimutagenic and

even leaf movement regulating agents that protect the organism that produces them

from the oxidative stress created by metabolism and their physical environment [294].

They also have antiviral, antibacterial (bactericidal, bacteriostatic), algicidal, plant

273

growth regulating, phytotoxic, antifungal, antiprotozoal, nematicidal, insecticidal,

antifeedant, and mammalian estrogenic, keratolytic, platelet aggregation inhibiting,

hypoglycemic, cytotoxic, and neurotoxic activities that may serve to protect the

organism that biosynthesizes them from competing, pathogenic, and herbivorous

organisms in their biological environment [295][296]. The diverse biological

functions of these phenolic acids suggest potential pharmacological activities.

11.1.5 Coumarins

&RXPDULQV RZH WKHLU FODVV QDPH WR µFRXPDURX¶ WKH YHUQDFXODU QDPH RI WKH WRQND

bean (Dipteryx odorata Willd., Fabaceae), from which coumarin itself was isolated in

1820 [297]. The primary site of synthesis of coumarins is suggested to be the young,

actively growing leaves, with stems and roots playing a comparatively minor role.

Coumarins belong to a group of compounds known as the benzopyrones, all of which

consist of a benzene ring joined to a pyrone (Figure 11.2). To this days, around 1,300

coumarins are known, with all of them being derivatives of 5,6-benzo-2- pirone (Į-

chromone) (with OH, OCH3 or CH3 substituents on the benzoic ring.). Coumarins

may also be found in nature in combination with sugars, as glycosides. As derivatives

of simple coumarins, other compounds are known, such as furanocoumarins, which

include a furanic ring, linear pyranocoumarins, angular pyranocoumarins, dimeric

coumarins, of which dicoumarol is an example and also furanochromones [298].

Historically, the ability of dicoumarol to inhibit blood clotting, that later led to the

development of the anticoagulant drug warfarin, was the first call to this class of

FRPSRXQGV¶ ELRORJLFDO SURSHUWLHV Coumarin is the parent molecule of warfarin,

274

which acts as a vitamin K antagonist. Warfarin is a clinically useful anticoagulant and

widely employed rodenticide. Several biological activities have been reported in

natural-occurring coumarins, from photo sensitizers to vasodilatation. Recently, the

interest has been given to synthetic derivatives of coumarins, such as fluorinated and

1-azo coumarins, which displayed moderate analgesia properties, and excellent anti-

inflammatory and anti-microbial activities [299].

Coumarins have a variety of bioactivities including anticoagulant, estrogenic, dermal,

photosensitizing, antimicrobial, vasodilator, molluscidal, antihelmintic, sedative,

hypnotic, analgesic and hypothermic activity [300]. Various coumarins have been

reported to possess anti-inflammatory activity as shown in carrageenan-induced

inflammation and cotton pellet granuloma tests [301][302]. There have been reports

on efficacies of pure coumarins as well as extracts containing them against Gram-

positive and Gram-negative bacteria as well as fungi [303]. The free OH group at C-6

in the coumarins nucleus has been found to be important for antifungal activity, while

the free hydroxyl group at C-7 is important for antibacterial activity [304]. The

bioactivities of phototoxic psoralens and dicoumaroul derivatives are well known and

several of these compounds are used in antipsoriatic and anticoagulant therapy [305].

Other than psoriasis, skin disease like cutaneous T-cell lymphoma, atopic dermatitis,

urticaria pigmentosa and lichen planus [306] are treated with the photochemotherapy

of linear furanocoumarins (psoralens). The most widely used compound is

xanthotoxin [307]. Bergapten is considered a valuable alternative for chemotherapy of

psoriasis, since its clinical efficacy is comparable to that of xanthotoxin, although

bergapten requires higher cumulative UVA doses.

275

The inherent fluorescent properties of many coumarins are a key factor in many

applications. Areas where coumarins are widely used include estimation of enzymatic

activity [308], labeling of proteins, antibodies, DNA and lipids, derivatising agents in

chromatography, etc. Coumarins have a wide variety of uses in industry, mainly due

to their strong fragrant odour [308]. Uses include that of a sweetener and fixative of

perfumes (e.g. 3,4-dihydrocoumarin), an enhancer of natural oils such as lavender, a

food additive in combination with vanillin, a flavour/odour stabilizer in tobaccos and

an odour masker in paints and rubbers. 6-Methylcoumarin is mainly used as a flavour

enhancer, and 7- hydroxycoumarin is mainly used in sunscreens.

11.2 Material and methods

Eight compounds that have been isolated in Chapter 11 were subjected to various

biological assays.

11.2.1 Antioxidant capacity assays

Samples were dissolved in dimethyl sulfoxide (DMSO, R&M) prior to assay at a

stock concentration of 1 mg/ml, and serial dilution was done accordingly to obtain a

good EC50 curve. Trolox (6-hydroxy-2,5,7, 8-tetramethylchromon-2-carboxylic acid,

Sigma-Aldrich), vitamin C (l-ascorbic acid), and quercetin were used as positive

control at a stock concentration of 1 mg/ml. Assay was performed using Thermo

Scientific Varioskan Flash microtiter plate reader, linked to a computer equipped with

(SkanIt Software 2.4.3). EC50 values were determined using Prism 5.00 software. At

276

least three independent tests were performed for each sample. Method is as explained

in Chapter 4, DPPH assay (section 4.2.1.2), ABTS assay (section 4.2.2.2), FRAP

DVVD\ VHFWLRQ ȕ-carotene bleaching assay (section 4.2.4.2) and SOD assay

(section 4.2.5.2)


Test samples and positive controls were dissolved in dimethyl sulfoxide (DMSO,

R&M) prior to assay at a stock concentration of 1 mg/ml, and serial dilution was done

according to the assay to obtain a good EC50 curve. Absorbance was recorded using

Thermo Scientific Varioskan Flash microtiter plate reader, linked to a computer

equipped with (SkanIt Software 2.4.3). Percentage inhibition of enzyme was

determined by comparison of rates of reaction of samples relative to blank sample

using the formula (E ± S)/ E × 100, where E is the activity of enzyme without test

sample and S is the activity of enzyme with test sample. The experiments were done

in triplicate. Method has been explained in Chapter 5, 5-LOX inhibition assay (section

5.4.2.2) and the peroxidase endpoint assay for COX-1 and COX-2 (section 5.5.3.2)


Test samples and galanthamine which was used as positive control was dissolved in

dimethyl sulfoxide (DMSO, R&M) prior to assay at a stock concentration of 1 mg/ml,

and serial dilution was done accordingly to obtain a good EC50 curve. The reaction

was monitored utilizing a 96-well microplate Thermo Scientific Varioskan Flash


277

Percentage inhibition of AChE was determined by comparison of rates of reaction of

samples relative to blank sample (ethanol in phosphate buffer pH = 8) using the

formula (E ± S)/ E × 100, where E is the activity of enzyme without test sample and S

is the activity of enzyme with test sample. The experiments were done in triplicate.

Method is explained in Chapter 6 (section 6.4.6.2).

11.2.4 Anticancer assay

All samples except syringic acid (insufficient yield) were tested for this activity

against human derived cell lines MDA 468 (ER-) breast carcinoma]. Samples were

prepared as 1 mg/ml stock solutions, dissolved in DMSO, and stored at 4 0C,

protected from light for a maximum period of 4 weeks. Serial drug dilutions were

prepared in medium immediately prior to each assay. Due to good activity exhibited,

scopoletin was also tested against HT-29, MCF-7 and HCT 116 cell lines. Method is

as explained in Chapter 9 section 9.3.2.

11.2.5 Antibacterial assay

Due to insufficient yield of compounds isolated, all samples were tested for the

antimicrobial effect only against S. aureus ATCC 11632. Samples was dissolved in

dimethyl sulfoxide (DMSO, R&M) prior to assay at a stock concentration of 1 mg/ml.

Method used is as explained previously, MIC assay (section 7.2.2.2), MBC assay

(section 7.2.3.2) and time-kill assay (section 7.2.4.2).

278

11.2.6 Antiparasitic assay

Due to insufficient yield of compounds isolated, scopoletin was tested for both assays

and only scoparone, (+)-catechin, lioxin and vomifoliol was tested for the

antileshmanial assay. Method is as explained in chapter 8, for antiparasitic assay

against Giardia intestinalis and Entamoeba histolytica (section 8.3.2.2.1) and

antileishmanial activity against Leishmania donovani promastigotes (section

8.3.2.2.2).

11.2.7 Formulation of scopoletin

Due to potent antioxidant, antibacterial and anti-inflammatory activity (to be

discussed in 11.8), scopoletin was formulated as a gel. Scopoletin (25 mg) was

prepared as a 0.5 % w/w preparation. Scopoletin was dissolved in few drops of carrier

solvent DMSO. Then orange oil is added to increase the penetration via skin. Pure

aloe vera gel was then added in sufficent amount to make up a total of 5 g of gel.


For section 11.2.1-11.2.3, concentration±response curves were calculated using the

Prism software package 5.00 for Windows, GraphPad Software, San Diego California

USA, www.graphpad.com (GraphPad, San Diego, USA) and data were reported as

mean and SD values obtained from a minimum of three determinations. Non linear

best fit was plotted with SD and 95% confidence interval. All data were expressed as

279

mean ± standard deviation. SD was computed but values were not shown in the

graphs via this software. For section 11.2.4-6 growth curve of bacteria/parasite/cell

were analysed using MS-Excel and data were reported as mean and SD values

obtained from a minimum of three determinations. All data were expressed as mean ±

standard deviation. Data were analyzed using one way Anova followed by Tukey test.

A significant difference was considered at the level of P < 0.01.

11.3 Results

11.3.1 Antioxidant capacity assays

The antioxidant capacity of the isolated compounds is shown in Table 11.1. In the

DPPH assay, the highest antioxidative action was exhibited by (+)-catechin (EC50

4.77±0.01 µg/ml) similar with positive control trolox (EC50 4.77±0.01 µg/ml),

followed by hyperin (EC50 4.89±0.02 µg/ml), cynaroside (EC50 7.89±0.03 µg/ml),

syringic acid (EC50 15.43±0.02 µg/ml) and scopoletin (EC50 36.80±0.01 µg/ml). Both

lioxin and vomifoliol had EC50 >100 µg/ml.

In the ABTS assay, the highest antioxidative action was exhibited by cynaroside

(EC50 0.26±0.02 µg/ml) followed by scopoletin (EC50 1.08±0.03 µg/ml), (+)-catechin

(EC50 5.03±0.06 µg/ml), syringic acid (EC50 7.38±0.02 µg/ml) hyperin (EC50

8.19±0.04 µg/ml), vomifoliol (EC50 34.89±0.04 µg/ml) and lioxin (EC50 83.22±0.03

µg/ml).

280

In the FRAP assay, antioxidant activity was calculated as Ferrous Equivalents, the

concentration of samples which produced an absorbance value equal to that of 1mM

FeSO4. The compounds absorbance equivalent to 1 mM FeSO4 was calculated from

equation of linearity of scopoletin (y=0.0203x+0.5259, r2=0.9837), scoparone

(y=0.0009x+0.1708, r2=0.5026), (+)-catechin (y=0.0141x+0.1442, r2=0.7415),

hyperin (y=0.0140x+0.2189, r2=0.8319), cynaroside (y=0.0156x+0.3277, r2=0.6009),

vomifoliol (y=0.0062x+0.0549, r2=0.6990), lioxin (y=0.0126x+0.0471, r2=0.8546),

syringic acid (y=0.0172x+0.2963, r2=0.6901). Total FRAP value was determined

from the absorbance value above using the standard Fe (II) calibration curve equation

(y=0.0105x+0.0136, r2=0.9817). Both scopoletin and (+)-catechin displayed

significantly lower FRAP value (49.00±0.64 µg/ml and 75.59±0.42 µg/ml

respectively) than positive control ascorbic acid and quercetin (347.00±0.23 µg/ml

and 86.00±0.24 µg/ml respectively).

In the ȕ-carotene decolourisation assay, the most potent activity was displayed by

vomifoliol (EC50 6.85±0.37 µg/ml), followed by lioxin (EC50 20.13±0.47 µg/ml),

hyperin (EC50 32.21±0.35 µg/ml), syringic acid (EC50 53.69±0.36 µg/ml), cynaroside

(EC50 56.38±0.38 µg/ml), (+)-catechin (EC50 83.22±0.56 µg/ml) and scopoletin (EC50

124.50±0.07 µg/ml). Only scoparone had EC50 of >100 µg/ml.

In the SOD assay, both hyperin and (+)-catechin has IC50 values significantly lower

(IC50 0.75±0.03 µg/ml and IC50 0.94±0.27 µg/ml respectively) than positive control

SOD enzyme (IC50 1.60±0.06 µg/ml). Cynaroside (IC50 1.87±0.03 µg/ml) had activity

similar to SOD, followed by scoparone (IC50 48.72±0.02 µg/ml) and lioxin (IC50

53.69±0.38 µg/ml). Scopoletin, vomifoliol and syringic acid had IC50 of >100 µg/ml.

281


The results are shown in Table 11.2 and Figure 11.3-11.5. In the 5-LOX inhibition

assay, all isolated compounds displayed significantly lower IC50 compared to positive

control NDGA. Both scoparone and scopoletin (IC50 0.20±0.01 µg/ml and 0.34±0.01

µg/ml respectively) had the most potent 5-LOX enzyme inhibition (Figure 11.3)

which was more than eightyfold compared to NDGA (IC50 29.21±0.05 µg/ml). This

was followed by syringic acid (IC50 1.38±0.03 µg/ml), hyperin (IC50 3.02±0.02

µg/ml), vomifoliol (IC50 8.72±0.02 µg/ml), (+)-catechin (IC50 16.10±0.03 µg/ml),

lioxin (IC50 17.79±0.03 µg/ml) and cynaroside (IC50 18.12±0.04 µg/ml) (Figure 11.4).

In the COX enzyme inhibition assay, only (+)-catechin and syringic acid showed

good enzyme inhibition while no enzyme inhibition was noticed with lioxin and all

other compounds had IC50 values >100 µg/ml. Compound (+)-catechin displayed

COX-1 selective activity with COX-1/COX-2 ratio of 0.14 with COX-1 enzyme IC50

of 12.08±0.02 µg/ml and COX-2 enzyme IC50 83.89±0.03 µg/ml (Figure 11.5).

Syringic acid had COX-1 enzyme inhibition at IC50 34.89±0.02 µg/ml with COX-2

inhibition at IC50 >100 µg/ml.


The anti-acetylcholinesterase values for isolated compounds are shown in Table 11.3

and Figure 11.6. Only 4 compounds showed moderate enzyme inhibition namely

syringic acid (IC50 29.53±0.19 µg/ml), scopoletin (IC50 51.00±0.02 µg/ml), scoparone

(IC50 86.58±0.05 µg/ml) and vomifoliol (IC50 96.64±0.09 µg/ml) as shown in Figure

282

Table 11.1: Antioxidant values of isolated compounds from Canarium patentinervium Miq.

Compounds ABTS assay, EC50 ȝJPO

DPPH assay, EC50 ȝJPO

FRAP assay, FRAP value

ȝJPO

ȕ-carotene bleaching assay,

EC50 ȝJPO

Superoxide dismutase assay, IC50

ȝJPO scopoletin 1.08±0.03 36.80±0.01. 49±0.64 124.5±0.07 >100

scoparone >100 >100 11492±0.16 >100 48.72±0.02

(+)-catechin 5.03±0.06 4.77±0.01A 75.59±0.42 83.22±0.56 0.94±0.27C

hyperin 8.19±0.04 4.89±0.02 791.96±0.52 32.21±0.35 1.87±0.03D

cynaroside 0.26±0.02 7.79±0.03 694.63±0.32 56.38±0.38 0.75±0.03C

vomifoliol 34.89±0.04 >100 1686.89±0.28 6.85±0.37 >100

lioxin 83.22±0.03 >100 830.67±0.53 20.13±0.47 53.69±0.38

syringic acid 7.38±0.02 15.43±0.02 594.03±0.41 53.69±0.36 >100

AA 1.57±0.06 1.83±0.01 345.15±0.23 NA NA

QC 0.98±0.03 2.78±0.02A 88.16±0.24 1.70±0.03B NA

TRO 0.65±0.02 4.77±0.01A 35.00±0.54 1.69±0.03B NA

SOD NA NA NA NA 1.60±0.06D

AA: ascorbic acid, QC:quercetin, TRO:trolox and SOD: superoxide dismutase Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same capital letter are not significantly different (P<0.05) according to Tukey multiple comparison test.

283

Table 11.2: Anti-inflammatory values of the isolated compound from Canarium patentinervium Miq.

Compounds Anti-inflammatory assay 5-LOX, IC50 ȝJPO



COX-1/COX-2 ratio

scopoletin 0.34±0.01 >100 >100

scoparone 0.20±0.01 >100 >100

(+)-catechin 16.1±0.03 12.08±0.02 83.89±0.03 0.14

hyperin 3.02±0.02 >100 >100

cynaroside 18.12±0.04 >100 >100

vomifoliol 8.72±0.02 >100 >100

lioxin 17.79±0.03 NO INH NO INH

syringic acid 1.38±0.03 34.89±0.02 >100

NDGA 29.21±0.05 NA NA NA

INDO NA 0.30±0.04 0.25±0.03 1.20

NDGA: nordihydroguairetic acid, INDO: indomethacin, NO INH: no inhibition noted Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

284

0 1 2 3 4 5

0

50

100scoparone

scopoletin

NDGA


% 5

-LO

X in

hibi

tion

Figure 11.3: 5-LOX inhibition by scopoletin and scoparone isolated from Canarium patentinervium Miq

285

0 5 10 15 20 25

0

50

100

lioxin

cynaroside

vomifoliol

hyperin

syringic acid

NDGA


% 5

-LO

X inhib

itio

n

Figure 11.4: 5-LOX inhibition by compounds isolated from Canarium patentinervium Miq

286

0 20 40 60 80 100

0

20

40

60

80

COX-1

COX-2

concentration(ug/ml)

% I

nhib

ition

by

CO

X

Figure 11.5: COX inhibition by (+)-catechin isolated from Canarium patentinervium Miq.

287

11.6. Rest of the compounds showed poor enzyme inhibition with IC50 values >100

µg/ml.

11.3.4 Anticancer assay

The anticancer activity of the compounds that were tested are shown in Table 11.4

and Figure 11.7a and 11.7b. Isolated compound with the highest yield scopoletin was

tested against four cell lines, two human breast cancer cell lines, MCF-7 (ER+) and

MDA 468 (ER-), and two colon cancer cell line, HCT 116 and HT-29. Scopoletin

exhibited potent growth inhibition of MDA 468 (GI50 0.09±0.25 µg/ml) (Figure

11.7a) and HT-29 (GI50 0.17±0.05 µg/ml) (Figure 11.7b) with significantly lower GI50

compared to positive control doxorubicin (GI50 0.69±0.06 µg/ml) for the HT-29 cell

line. Cell line MCF-7 and HCT 116 were less sensitive to scopoletin with GI50 values

>2 µg/ml. Scoparone, (+)-catechin, hyperin, cynaroside, vomifoliol and lioxin were

tested only against MDA 468 cell line and all having GI50 values >2 µg/ml.

11.3.5 Antibacterial assay

The antibacterial assay results are shown in Table 11.5 and Figure 11.8.

Staphylococcus aureus ATCC 11632 was sensitive against all compounds tested.

Scopoletin had the best activity with MIC 25.00±0.00 µg/ml and MBC of 50.00±0.00

µg/ml with complete bacterial kill at 50.00±0.00 µg/ml (Figure 11.8). Scoparone,

Hyperin, cynaroside and syringic acid had bactericidal activity at 100.00±0.00 µg/ml.

288

Table 11.3: Anti- acetylcholinesterase values for isolated compounds from Canarium patentinervium Miq.

Compounds Anti-acetylcholinesterase assay, IC50ȝJPO scopoletin 51.00±0.02

scoparone 86.58±0.05

(+)-catechin >100

hyperin >100

cynaroside >100

vomifoliol 96.64±0.09

lioxin >100

syringic acid 29.53±0.19

galantamine 0.77±0.09

Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD.

Values with the same letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

289

0 20 40 60 80 100

0

20

40

60

80

100syringic acid

vomifoliol

scoparone

scopoletin


% in

hibi

tion

of A

ChE

Figure 11.6: AChE inhibition by isolated compounds from Canarium patentinervium Miq.

290

Table 11.4: Growth inhibition of human cancer cell lines by isolated compounds from Canarium patentinervium Miq.

Compounds Cell line/ GI50 (µg/ml)

MDA 468 HT-29 MCF-7 HCT 116

scopoletin 0.09±0.25 0.17±0.05 >2 >2

scoparone >2 nt nt nt

(+)-catechin >2 nt nt nt

hyperin >2 nt nt nt

cynaroside >2 nt nt nt

vomifoliol >2 nt nt nt

lioxin >2 nt nt nt

doxorubicin 0.04±0.12 0.69±0.06 0.38±0.28 0.65±0.18

Nt = not tested, Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same letter are not significantly different (P<0.01) according to Tukey multiple comparison test.

291

Figure 11.7a: Effect of scopoletin isolated compounds from Canarium patentinervium Miq. against growth of breast cancer cell line MDA 468

(GI50 = 0.09 µg/ml)

0

0.5

1

1.5

2

2.5

control 0.01 0.02 0.1 0.2 1 2

OD

55

0 n

m


scopoletin

T zero

292

Figure 11.7b: Effect of scopoletin isolated compounds from Canarium patentinervium Miq. against growth of colon carcinoma cell line HT-29

(GI50 = 0.17 µg/ml)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

control 0.01 0.05 0.1 0.5 1 5

OD

55

0 n

m


scopoletin

T zero

293

11.3.6 Antiparasitic assay

The antiparasitic activity of the compounds tested is shown in Table 11.6. Compound

scopoletin was tested against all three parasite amd it was more potent against

Leishmania donovani (IC50 163.30±0.32 µg/ml) and MIC of >200 µg/ml for both

Giardia intestinalis and Entamoeba histolytica. Sensitivity of Leishmania donovani is

then followed by with the presence of lioxin (IC50 211.48±0.32 µg/ml), vomifoliol

(IC50 302.80±0.33 µg/ml), scoparone (IC50 329.90±0.32 µg/ml) and (+)-catechin (IC50

478.93±0.28 µg/ml).

11.4 Discussion

Of the eight isolated compounds, five were phenolics (hyperin, cynaroside, (+)-

catechin, lioxin and syringic acid), two were coumarins (scopoletin and scoparone)

and one was a norsesquiterpene with a cyclohexenone ring (vomifoliol). Among the

phenolics, (+)-catechin is a flavanol, hyperin is a flavonol with a glucosidic moiety

and cynaroside is a flavone with a glycosidic moiety and two were benzoic acid

derivatives. The basic skeleton of these groups are shown in Figure 11.2.

Scopoletin possesses interesting activities, in particular, vasorelaxant [309],

antioxidant [310],antimicrobial [311], anti-inflammatory [312], anti-pyretic [313],

anti-platelet aggregation [314] and anti-diabetes mellitus properties [252]. In addition,

294

Table 11.5: MIC, MBC and MBC/MIC ratio for isolated compounds from Canarium patentinervium Miq. against Staphylococcus aureus ATCC 11632

Compounds Concentration (µg/ml)

MBC/MIC ratio MIC MBC

scopoletin 25.00±0.00 50.00±0.00 2 (+)

scoparone 50.00±0.00 100.00±0.00 2 (+)

(+)-catechin 50.00±0.00 >100 nd

hyperin 50.00±0.00 100.00±0.00 2 (+)

cynaroside 50.00±0.00 100.00±0.00 2 (+)

vomifoliol 100.00±0.00 >100 nd

lioxin 100.00±0.00 >100 nd

syringic acid 50.00±0.00 100.00±0.00 2 (+)

ND: not determined

295

Figure 11.8: Time-kill plot for MSSA ATCC 11632 in presence of scopoletin isolated from Canarium patentinervium Miq.

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Bac

teri

al g

row

th (

incr

ease

in O

D)

Time (hr)

0 ug/ml 6.25ug/ml 12.5ug/ml 25ug/ml 50ug/ml

296

Table 11.6: Antiparasitic values of isolated compounds from Canarium patentinervium Miq. against

Leishmania donovani (AG 83) promastigotes and

MIC values against Giardia intestinalis and Entamoeba histolytica growing in vitro.

Compounds

IC50 ( (µg/ml) MIC (µg/ml)

Leishmania donovani Giardia intestinalis Entamoeba histolytica

scopoletin 163.30±0.32 >200 >200

scoparone 329.90±0.32 nt nt

(+)-catechin 478.93±0.28 nt nt

vomifoliol 302.80±0.33 nt nt

lioxin 211.48±0.32 nt nt

Data were obtained from three independent experiments, each performed in triplicates (n=9) and represented as mean ± SD. Values with the same letter are not significantly different (P<0.01) according to Tukey multiple comparison test. nt: not tested

297

it exerted neuroprotective [315] and hypotensive [316] activities in addition to

applications in cardiovascular disease [252], antitumor [317], anti-proliferation and

antithyroid [318] treatment. Scoparone has been studied for its potential

pharmacological properties including immunosuppression [319] and vasorelaxation

[320]. Compound (+)-catechin is reported to induce longevity in the nematode worm

Caenorhabditis elegans [321], reduces atherosclerotic lesion development in mice

[322], have opposite effects on glycogen metabolism in isolated rat hepatocytes [323],

inhibits intestinal tumor formation in mice [324], inhibits the oxidation of low density

lipoprotein [325] and as an antioxidant [326]. Cynaroside has been reported for

moderate inhibition of enzymes for the synthesis of thromboxane B2 and leukotriene

B4 as well as hydrogen peroxide scavenging activity, scavenge reactive oxygen and

nitrogen species [327] to chelate transition metals, antibacterial [328], antiviral [329]

and antifungal [330] activity.

Hyperin has been found to have antioxidant activity [331], inhibition of lipid

peroxidation in rat liver microsomes [332] and protective effects to PC12 cells against

cytotoxicity induced by hydrogen peroxide and tert-butyl hydroperoxide [333]. Lioxin

had been reported to exert strong antioxidant (oral protectant) [334], powerful wound

healing properties [335], protective effects against DNA damage [336] as well as

antimutagenic [337] and immunostimulating [338] properties. Syringic acid has

shown to have antioxidant, antibacterial [339] and hepatoprotective [340] activities.

Vomifoliol have been reported to have weak DPPH activity [341] and anti-

inflammatory activity by inhibition of TNF-Į [341] and plays an important role as an

endogenous regulator of the stomatal aperture [262].

298

11.4.1 Antioxidant activity

The antioxidant mechanisms of flavonoids comprise free radical scavenging, but also

enzyme inhibition/induction, metal chelation, interaction with receptors and

modulation of gene expression [342]. The direct scavenging of radicals is believed to

occur in cells, but, to avoid the interference of other mechanisms, this work was

developed using a cell-free system. The past few decades of structure-activity

relationships research have generated several consistent lines of evidence supporting

the role of specific structural components as requisites for antioxidant activity. The

antioxidant activity of flavonoids and their metabolites in vitro depends upon the

arrangement of functional groups about the nuclear structure, namely ring A, B and C

[343]. Consistent with most polyphenolic antioxidants, both the configuration and

total number of hydroxyl groups substantially influence several mechanisms of

antioxidant activity. The B-ring hydroxyl configuration is the most significant

determinant of scavenging of ROS and RNS. A-ring substitution correlates little with

antioxidant activity [344].

Some characteristics of flavonoids are important to the antioxidant capacity according

to the famous three Bors criteria [344] (Figure 11.9): (i) o-dihydroxy structure in the

B ring LHD¶¶-catechol structure, which confers higher stability to the radical form

and participates in electron delocalization ; (ii) C2-C3 double bond in conjugation with

a 4-oxo function in the C ring, responsible for electron delocalization of the aromatic

nucleus; (iii) 3- and 5-OH groups with 4-oxo function in the A and C rings, required

for maximum radical scavenging potential. Moreover, an additional criterion could be

added: (iv) In the absence of o-dihydroxy structure in the B ring, hydroxyl

299

substituents in a catechol structure on the A ring are able to compensate and become a

larger determinant of flavonoid antiradical activity.

Figure 11.9: Structural features of flavonoids with high antioxidant activity.

Of the three flavonoids, (+)-catechin and hyperin exhibited the most consistent free

radical scavenging capability across the five antioxidant assay (Table 11.1). Good

correlation was found between FRAP values and hydroxyl groups and especially the

catechol moiety and 3-OH [345], explaning the best activity shown by (+)-catechin

(FRAP value 75.59±0.42 µg/ml). Results in the ABTS and DPPH assay were

relatively similar as they have the same mechanism of action [53]. Sawai et al. [346]

have identified the antioxidant mechanism using NMR analytical approaches to

identify the antioxidative molecular mechanism in DPPH assay and found that the B

ring of (+)-catechin has been changed to a quinone structure as shown in Figure

11.10.

300

Figure 11.10: (+)-catechin and the corresponding fully oxidized ortho-quinone product

Flavanols, namely here (+)-catechin and flavonol (hyperin) are more effective free

radical scavengers than flavones (cynaroside) [343], which may be ascribed to the

greater number of hydroxyl groups and 3-OH to the former two. Plumb and co-

workers [347] reported that the antioxidant properties of flavonol glycosides from tea

decreased as the number of glycosidic moieties increased. In addition, glycosylation

of flavonoids mostly decreases their antioxidant activity. Multiple A-ring methoxy

groups as seen in cynaroside also reverse the positive effect of a B-ring catechol

[344].

+RZHYHU LQ WKH ȕ-carotene bleaching assay, which involves lipid peroxidation via

hydrogen atom transfer (HAT method- section 4.5), hyperin and cynaroside

outweighed activity of (+)-catechin. Flavonoids with a C2±C3 double bond in

conjugation with a 4-carbonyl group has been known to exhibit lower EC50 values

(indicative of stronger antioxidant activity) in a microsomal system compared to those

with saturated heterocycles [344] explaining the lower EC50 values of cynaroside

(56.38±0.38 µg/ml) and hyperin (32.21±0.35 µg/ml) LQ WKH ȕ-carotene bleaching

assay. However, hyperin has a lower EC50 than cynaroside in this assay as the C-3

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position is an excellent choice for substitution to give the flavonoid an optimum

lipophilicity [348].

Between both glycosides, hyperin was superior to cynaroside in all five assays (Table

11.1) as C-glycosylation in the A-ring (seen in A ring of cynaroside) results in greater

diminution of activity than 3-glycosylation (seen in hyperin) in the heterocycle [349].

Studies have also shown that O-glycosylation at carbon 7, but not carbon 3, weakens

the antioxidant effect of flavonoids in rat mitochondria [349]. O-glycosylation

interferes with the coplanarity of the B-ring with the rest of the flavonoid and the

ability to delocalize electrons [350]. Though glycosides are usually weaker

antioxidants than aglycones, bioavailability is sometimes enhanced by a glucose

moiety [349]. The SOD assay involves flavonoids having SOD-like activity and

inhibition of XOD that produces the free radicals. Hyperin and (+)-catechin have

significantly lower IC50 (0.75±0.03 µg/ml and 0.94±0.27 µg/ml respectively)

compared to SOD enzyme. This is due to the structure arrangement of hyperin as the

planar flavonoid core (double bond between C-2 and C-3) and the presence of free

hydroxyl groups at C-5 and C-7 are important prerequisites for this activity [344]. (+)-

catechin has slightly less activity compared to hyperin as it lacks the double bond

between C-2 and C-3).

Phenolic acids have been investigated for the radical scavenging activity via ABTS

assay by Rice- Evans [351]. Structure-activity comparisons suggest that antioxidant

activity of phenolic acids depends on the number and orientation of hydroxyl groups

relative to the electron-withdrawing CO2H, CH2CO2H, or (CH)2CO2CH functional

302

group [39]. It can be seen that syringic acid has more electron withdrawing groups

compared to lioxin confirming the lower EC50 in ABTS assay (7.38±0.02 µg/ml).

Between both coumarins, scopoletin exhibited potent antioxidant activity compared to

scoparone. It had significantly lower EC50 values in ABTS (EC50 1.08±0.03 µg/ml)

compared to ascorbic acid (EC50 1.57±0.06 µg/ml) and lower values in FRAP assay

(FRAP value 49.00±0.64 µg/ml) than quercetin (FRAP value 88.16±0.24 µg/ml) and

ascorbic acid (FRAP value 345.00±0.23 µg/ml). The result is consistent with a

previous report that the scavenging capacity was higher for coumarin bearing OH

groups than the methoxy-substituted derivatives [352] as in scoparone. In the SOD

assay, the SOD-like activity and inhibition of XOD that produces the free radicals

seemed to be better with scoparone (IC50 48.72±0.02 µg/ml) than scopoletin (IC50

>100 µg/ml). Reports have stated that a methoxy substituted for 6- OH diminishes

inhibition potency against XOD [352] as in the result of both coumarins here. This

further enhances the hypothesis that the H atom of the 6-hydroxyl plays a more

significant role than the O atom. It also can be observed that perhaps the 7-methoxy

group in scoparone increases the inhibition activity.

Not much biological data is available on vomifoliol except weak DPPH activity and

anti-inflammatory activity by inhibition of TNF-Į [341]. However in this study,

though consistent low DPPH activity is seen (EC50 >100 µg/ml), moderate activity in

noted in ABTS (EC50 JPODQGSRWHQWȕ-carotene bleaching activity was

noted (EC50 6.85±0.37 µg/ml).

303

11.4.2 Anti-inflammatory activity

All isolated compounds exhibited significantly lower 5-LOX inhibition than positive

NDGA (IC50 29.21±0.05 µg/ml). Among the five phenolics, syringic acid and hyperin

had the most potent 5-LOX inhibitory activity (IC50 1.38±0.03 µg/ml and 3.02±0.02

µg/ml respectively). Hydroxylations at 5, 7, and 4' have also shown to enhance overall

anti-inflammatory activity in flavonoids [102]. Flavonols were found to be stronger

inhibitors of 5-LOX than flavones [353], as established in this study with hyperin

(IC50 3.02±0.02 µg/ml) being superior to cynaroside (IC50 18.12±0.04 µg/ml).

Although it is difficult to establish structural-activity relationships due to their

varieties of chemical structures, these inhibitory activities against 5-LOX could

explain, at least in part, the anti-inflammatory/ antiallergic activities of flavonoids. No

clinical data showing the relation of flavonoid intake and incidence (severity) of

inflammatory disorders such as RA and AD was available, although several studies

demonstrated some inverse correlation of flavonoid intake and incident rate of

cardiovascular failure [354].

Lipoxygenases (LOXs) also function as prooxidant enzymes that catalyze enzymatic

lipid peroxidation. Additionally, they are a source of free radicals initiating

nonenzymatic lipid peroxidation and other oxidative processes involved in the

formation of atherosclerotic lesions [354]. Consequently, inhibition of LOXs may

contribute to the universal antioxidant activities of flavonoids. Planar flavonoid

structure with delocalized electrons achieved by the C2-C3 double bond seems to be a

prerequisite for inhibitory activity. The following structural features were found to

enhance the inhibitory potency: (i) presence of a catechol moiety in the B or A ring,

304

(ii) 4-carbonyl group in the C ring [344], thus confirming the results of this study with

hyperin having more 5-LOX inhibition than (+)-catechin.

However (+)-catechin had a more comprehensive anti-inflammatory activity with dual

inhibition of 5-LOX (IC50 16.10±0.03 µg/ml) and COX (COX-1; IC50 12.08±0.02

µg/ml, COX-2; IC50 83.89±0.03 µg/ml). (+)-catechin displayed COX-1 selective

activity with the COX-1/COX-2 ratio <1 and this confirms previous findings [107].

Benzoic acid derivatives, syringic acid exhibited potent 5-LOX inhibition (IC50

1.38±0.03 µg/ml) and moderate COX-1 inhibition (IC50 34.89±0.02 µg/ml) compared

to lioxin that had good 5-LOX inhibition (IC50 17.79±0.03 µg/ml) but no COX

inhibition. Consideration of the structure-activity relationship of phenolic acids on the

anti-inflammatory action revealed that both C-4 hydroxy and C-3 methoxy radicals of

the benzoic acid derivatives play an important role in anti-inflammatory activities

[355]. However the presence of extra methoxy group at C-5 seemed to drastically

increase the activity as seen in syringic acid. One study reported potent COX-2

inhibition of syringic acid (IC50 0.40 µg/ml) using cell based study [356] but we

demonstrated poor COX-2 inhibition in this study.

Both coumarins scopoletin and scoparone were potent inhibitors of 5-LOX recording

lowest IC50 values (IC50 0.34±0.01 µg/ml and 0.20±0.01 µg/ml respectively).

Scoparone has slightly lower IC50 than scopoletin as the SAR findings has reported

that the chloro and methoxy substitution in coumarin ring showed increase anti 5-

LOX activity [357]. Cyclohexenone derivatives are well known lead molecules for the

treatment of inflammation and autoimmune diseases [358]. The anti-inflamatory

activity previously reported for vomifoliol were by inhibition of TNF-Į [341]. In this

305

study, vomifoliol has exhibited potent 5-LOX inhibition (IC50 8.72±0.02 µg/ml) and

poor COX inhibition.

11.4.3 Anti-acetylcholinesterase activity Only the scopoletin, scoparone, vomifoliol and syringic acid showed AChE inhibition

at IC50 <100 µg/ml. The studies have also shown that naturally occurring as well as

the chemically synthesised coumarin analogs exhibit potent AChE inhibitory activity

[359]. Coumarin ring seems to be essential for the optimal activity and its replacement

with related structural moiety such as chromone is associated with loss of AChE

inhibitory activity. The substituents at coumarin moiety particularly at 6th and 7th

position also influence the activity in a significant manner. The presence of electron-

donating groups such as ±OCH3, ± OH, and ±NH2 increase the activity and it has

been generally attributed to increase in lipophilicity of compounds [360]. This might

explain good activity of scopoletin (IC50 51.00±0.02 µg/ml) and scoparone (IC50

86.58±0.05 µg/ml) as both have electron donating groups at position 6th and 7th. The

presence of bulkier substituents at 6th and 7th positions of the coumarin is associated

with significant loss in AChE inhibitory activity indicating the critical role of

electronic as well as steric effects in influencing the AChE inhibitory activity [360],

this explains why scoparone has higher IC50 due to it having bulkier two methoxy

groups.

Up to date, quite a lot of studies have reported affirmative effects of phenolics in

neurodegenerative diseases depending upon their antioxidative properties [361].

However, there has been a small number of data on AChE inhibitory activities of

306

phenolic compounds. Among the phenolics isolated in this study only syringic acid

exhibited good AChE inhibition (IC50 29.53±0.19 µg/ml), lowest of all compounds

tested. The literature concerning the role of phenolic acids and their derivatives in the

neuroprotection of the CNS is, however, still incomplete. Thus the actual mechanism

of inhibition needs to be investigated. Cyclohexenone derivatives, vomifoliol had

moderate AChE inhibition (IC50 96.64±0.09 µg/ml), which is to the best of our

knowledge the first to be reported.

11.4.4 Antiparasitic activity

Only scopoletin exhibited moderate antileishmanial activity (IC50 163.30±0.32 µg/ml)

followed by lioxin, vomifoliol, scoparone and (+)-catechin. Poor activity of scopoletin

was noted against Giardia intestinalis and Entamoeba histolytica while the others

were not tested against them. Comparing the anti AChE and antiparasitic activity, it

can be deduced that same compounds seemed active in both assays. Choline is the

precursor of phosphatidylcholine (PC), a main component of Leishmania

promastigote membranes [362]. Therefore, inhibition of choline formation may de-

crease Leishmania survival. This hypothesis can be tested by using inhibitors of the

acetylcholinesterase enzyme (AChE), which catalyzes the hydrolysis of acetylcholine

to choline and acetic acid, as leishmanicidal compounds. This may identify another

mechanism of action for leishmanicidal activity [363].

The lactone groups present in coumarins are also present in the structures of

Annonaceous acetogenins that show leishmanicidal activity [364]. The AChE inhibi-

tory activity of a previous study on scoparone indicates a possible action mechanism

307

E\ GLVUXSWLQJ WKH YLDELOLW\ RI OHLVKPDQLD¶V FHOO PHPEUDQHV. There has been a

hypothesis that the coumarin and scoparone have mechanism of action acting on the

same pathway as above compounds, resulting in a net negative effects on choline

uptake by the parasite [363]. Only flavonoid (+)-catechin was tested in this study.

Flavonoids however, are able to inhibit acetylcholine hydrolysis and interfere in the

PC due to their low concentration of choline precursor from the host. Nevertheless,

the leishmanicidal activity of these compounds may be related to their ability to

chelate iron (Fe), depriving this essential nutrient from the intracellular forms [365].

11.4.5 Anticancer activity

In this study, scopoletin displayed potent anticancer effect against breast cancer cell

line MDA 468 (GI50 0.09±0.25 µg/ml) and colorectal cancer cell line HT-29 (GI50

0.17±0.05 µg/ml), the latter being more significant that positive control doxorubicin

(GI50 0.69±0.06 µg/ml). Previously studies on scopoletin have shown it to inhibit the

cell proliferation by inducing cell cycle arrest and the increase of apoptosis in human

prostate tumor cells [366], and it induced apoptosis of HL-60 cells [367]. The ability

of scopoletin have been said to induce programmed cell death in leukemic cells

representing a novel and important aspect of the discussion of the anti-tumoral effects

of various plants containing scopoletin [367]. In this study, scopoletin has shown

potent growth inhibition of these two cell lines warranting further study.

308

11.4.6 Antibacterial activity

All isolated compounds tested against Staphylococcus aureus ATCC 11632 showed

bacterial growth inhibition. Scopoletin, scoparone, hyperin, cynaroside and syringic

acid had bactericidal effect <100 µg/ml. Only scopoletin had bactericidal effect and

complete kill at MBC 50.00 µg/ml. The antibacterial properties of coumarins were

first recognised in 1945 when an investigation with dicoumarol was found to inhibit

the growth of several strains of bacteria. All the methods reported in the study of the

antibacterial activity of coumarins were disc diffusion methods [368]. It has then been

suggested that coumarins possess antibacterial activity act selectively against Gram-

positive microorganisms [368]. Free hydroxyl group at position 7 has been shown to

be important for antibacterial activity [298] as confirming the activity of scopoletin

that has free 7-OH.

11.4.7 Formulation of scopoletin as a gel

Previously study by Seon et al. [369] have demonstrated scopoletin to inhibit

tyrosinase kinase with an IC50 of 10.2 µg/ml suggecting its possible use in skin aging

and preventive activities. Biosynthesis of melanin in animals is normally initiated

through the oxidation of tyrosine into DOPA by tyrosinase which is the rate limiting

enzyme in this pathway. The production of abnormal pigmentation such as melasma,

freckles, senile lentigines, and other forms of melanin hyperpigmentation could be a

serious aesthetic problem [369]. In this study scopoletin was found to exhibit potent

antioxidant, anti-inflammatory and antibacterial properties. Scopoletin has been

studied to have good pharmacokinetics data with good absorption in the stomach and

309

colon of rats [370]. Reported physical properties of scopoletin which passes the

Lipinsky rule (mass < 500, log < 5, donor count < 5, and acceptor count < 10), for

possible lead compound in drug discovery [371] and, in agreement with its potent

antioxidant power, good anti-inflammatory and antibacterial activity enables it to be

considered for a formulation for further test and development.

Transdermal drug delivery offers the following advantages over oral administration:

(1) peak and valley levels in the serum are avoided; (2) first-pass metabolism is

avoided and the skin metabolism is relatively low; (3) less frequent dosing regimens

is needed due to the maintenance and longer sustainability of zero-order drug delivery

and (4) less inter-subject variability occurs [372]. Other advantages listed include

aspects such as the accessibility of the skin; a relatively large surface area for

absorption and the fact that it is non-invasive make it more patient compliant [373].

Thus, an external formulation suitable for cosmetic use (skin hyperpigmentation, acne

and wrinkle protection) and for medicinal use as an anti-inflammatory gel was

derived. The formulation was prepared with aloe vera gel as a base due to the well

known skin and medicinal properties of aloe [374]. Orange oil was used as studies

have shown the limonene (moneterpene) content has a high ability to enhance in vitro

percutaneous transport greater than conventional lipophilic penetration enhancing

FRPSRXQGV HJ 6SDQ Į-bisabolol, oleic acid) and hydrophilic penetration

enhancing compounds (e.g., ethanol, polyethylene glycol 600, 1,8-cineole) [375].

Further studies on the efficacy of the gel and penetration test are required.

310

11.5 Conclusion

For the first time, eight compounds (scopoletin, scoparone, (+)-catechin, lioxin,

cynaroside, hyperin, vomifoliol and syringic acid) were isolated from Canarium

patentinervium Miq. Three of these compounds (vomifoliol, lioxin and syringic acid)

were isolated for the first time from the genus Canarium. These compounds were then

subjected to a range of biological assays, all reported for the first time from this plant.

Canarium patentinervium Miq. is a rare plant from the family of Burseraceae and

genus Canarium found in Asia Pacific region previously recorded for its usage in

wound healing by the indigenous people of Malaysia. One of the objectives of this

study was to ascertain the pharmacological and phytochemical aspect for the

ethnopharmacological usage. Wound healing involves manifold inflammatory

processes of which notably the massive release of leukotrienes from arachidonic acid

via the 5-lipoxygenase pathway (5-LOX) and the generation of nitric oxide (NO) from

inducible nitric oxide synthase (iNOS). Of note, nitric oxide (NO) is a free radical,

and the generation of cytokines involves a reactive oxygen species (ROS) outburst.

Therefore, agents able to block the enzymatic activity of 5-LOX and to scavenge free

radical are of immense interest against inflammatory conditions which englobe not

only epidermal insults but also neurodegeneration and obesity.

Cells produce superoxide anion (O2í), peroxide anion (HO2

í), and hydroxyl ion (HOí)

as part of the physiological aerobic metabolism which are quickly scavenged by

cytoplasmic antioxidant defense system. However, in the event of ageing or

pathologies, the antioxidant defense system is overwhelmed, and cells suffer massive

311

oxidative stress leading eventually to carcinogenesis or apoptosis. In fact, oxidative

stress is the main causative factor for cholinergic and dopaminergic neurons apoptosis

hence $' DQG 3DUNLQVRQ¶V GLVHDVH 3'. In addition, there is a growing body of

evidence that point to the fact that 5-LOX inhibitors are of immense therapeutic

values. The various isolated secondary metabolites clearly demonstrate various potent

biological activities that substantiate the ethnopharmacological claim.

Given the aforementioned evidence it is tempting to speculate that Canarium

patentinervium Miq. represents an exciting scaffold from which to develop leads for

treatment of neurodegenerative diseases.

312

CHAPTER 12

GENERAL CONCLUSIONS

The objectives of the study were to:

i. Extraction of leaves and barks of plant with solvents of different

polarity to obtain crude extract with solvents of different polarity.

ii. Investigation of the possible secondary metabolite types present in

the crude extracts of leaves and barks with qualitative

phytochemical analysis.

iii. Isolation and identification of the chemical compounds responsible

for the antibacterial and antioxidant activity in the most active

extract through bioassay-guided fractionation using liquid-liquid

extraction or solvent partioning, column chromatography, thin

layer chromatography (TLC) and nuclear magnetic resonance

(NMR).

iv. Investigation of the antioxidant activity of the crude extracts of

both leaves and barks using the 2,2-diphenyl-1-picrylhydrazyl

(DPPH) radical scavenging assay, ferric reducing ability of plasma

)5$3DVVD\¶-Azino-bis(3-ethyl-14 benzthiazoline-6-sulfonic

DFLG $%76DVVD\ ȕ-carotene bleaching assay and superoxide

dismutase assay.

313

v. Investigation of the anti-inflammatory potential of the crude

extracts against 5-lipoxygenase and cycloxygenase systems in

vitro.

vi. Investigation of the anti AChE potential of the crude extracts in

vitro.

vii. Investigation of the in vitro antibacterial activity of the crude

extracts of leaves and barks using the disc diffusion assay and

determination of minimum inhibitory concentration, minimum

bactericidal concentration and death kill rate against four ATCC

bacterial strains namely Gram-positive bacteria Staphylococcus

aureus, Bacillus cereus, methicillin-resistant Staphylococcus

aureus and Gram-negative Pseudomonas aeruginosa and ten

clinical strain bacteria.

viii. Investigation of the in vitro antifungal activity of the crude extracts

of leaves and barks using the disc diffusion assay against three

clinical strain fungi namely, Candida parapsilosis, Candida

glabrata and Candida albicans.

ix. Investigation of the anticancer activity of the crude extracts of the

leaves and barks against human derived cell lines [HCT 116, colon

cancer cell line and MCF-7 (ER+), MDA 468 (ER-) breast

carcinoma] using MTT assay.

x. Investigation of the in vitro anti-parasitic activity of the crude

extracts of leaves and barks against Leishmania. donovani

promastigotes, Entamoeba histolytica and Giardia intestinalis.

314

xi. Bioactivity screening of all isolated compounds using the same

bioassay as mentioned in iii-ix.

The following appropriate conclusions were drawn based on the experimental data

obtained.

i. Extraction of samples

Extraction was done with maceration with solvents of increasing

polarity.The yield for the hexane, chloroform and ethanol extract of leaves

were 1.25 %, 1.11 % and 6.45 % respectively. The yield for the hexane,

chloroform and ethanol extract of barks was 1.04 %, 0.4 % and 2.61 %.

Crude extracts were kept at -20oC until further use.

ii. Phytochemical analysis

Phytochemical analysis of Canarium patentinervium Miq. revealed

presence of tannins and flavonoids in the ethanol extract of leaves and

barks. Steroids were detected in the ethanol extract of leaves and all tested

bark extracts. Results from our phytochemical analysis revealed that the

ethanol extract of leaves and barks of Canarium patentinervium Miq.

accumulate substantial amounts of flavonoids and tannins which could be

possibly result in antioxidant or antibacterial activity.

iii. Isolation of biologically active compounds

Eight compounds were isolated for the first time from Canarium

patentinervium Miq. using various isolation techniques such as TLC, CC,

HPLC and identified with NMR method. Compounds isolated scopoletin,

315

scoparone, (+)-catechin, hyperin, cynaroside, lioxin and syringic acid were

phenolics while vomifoliol was a norsesquiterpene with a cyclohexenone

ring. Lioxin, syringic acid and vomifoliol were isolated from this genus

Canarium for the first time.

iv. Antioxidant activity

The ethanol extract of leaves and barks displayed superior antioxidant

capacities. The EC50 values of the samples were consistently low in SET

methods (ABTS, DPPH and FRAP assay) superior to standard as opposed

WR +$7 PHWKRG ȕ-carotene bleaching assay). The present study

demonstrates that Canarium patentinervium Miq. is a potent source of

antioxidants that exhibits its antioxidant activity predominantly via the

SET method.

Compounds (+)-catechin and hyperin exhibited the most consistent free

radical scavenging capability across the five antioxidant assay. Flavanols,

namely here (+)-catechin and flavonol (hyperin) are more effective free

radical scavengers than flavones (cynaroside). Hyperin and (+)-catechin

have significantly lower IC50 (0.75±0.03 µg/ml and 0.94±0.27 µg/ml

respectively) compared to SOD enzyme. Between both coumarins,

scopoletin exhibited potent antioxidant activity compared to scoparone. It

had significantly lower IC50 values in ABTS (IC50 1.08±0.03 µg/ml)

compared to ascorbic acid (IC50 1.54±0.03 µg/ml) and lower values in

FRAP assay (IC50 49.00±0.64 µg/ml) than quercetin (IC50 86.00±0.24

µg/ml) and ascorbic acid (IC50 347.00±0.23 µg/ml). Vomifoliol displayed

316

low DPPH activity (IC50 >100 µg/ml), moderate activity is noted in ABTS

(IC50 JPO DQG SRWHQW ȕ-carotene bleaching activity was

noted (IC50 6.85±0.37 µg/ml).

v. Anti-inflammatory activity

In view of dual inhibition of 5-LOX and COX activity, the ethanol extract

of leaves seemed to have moderate inhibition against 5-LOX and good

inhibition against COX enzymes (selective COX-2). Chloroform extract of

the bark though had good 5-LOX inhibition but did not have activity

DJDLQVW&2;HQ]\PHVEHORZȝJPO

In the 5-LOX inhibition assay, all isolated compounds displayed

significantly lower IC50 compared to positive control NDGA. Both

scoparone and scopoletin (IC50 0.20±0.01 µg/ml and 0.34±0.01 µg/ml

respectively) had the most potent 5-LOX enzyme inhibition (Figure 11.2)

which was more than eightyfold compared to NDGA (IC50 29.19±0.02

µg/ml). This was followed by syringic acid (IC50 1.38±0.03 µg/ml),

hyperin (IC50 3.02±0.02 µg/ml), vomifoliol (IC50 8.72±0.02 µg/ml), (+)-

catechin (IC50 16.10±0.03 µg/ml), lioxin (IC50 17.79±0.03 µg/ml) and

cynaroside (IC50 18.12±0.04 µg/ml).

vi. Anti AChE activity

Chloroform extract of the barks displayed the best activity (IC50 =

ȝJPODVRSSRVHGWRJDODQWKDPLQH,&50 ȝJPO

317

The ethanol extract of barks and leaves follow through with IC50 =

ȝJPO DQG ,&50 ȝJPO UHVSHFWLYHO\+H[DQH

extracts of bark and leaves and the chloroform extract of leaves had the

lowest enzyme inhibition activity (IC50 ȝJPO ,&

ȝJPO DQG ,&50 ȝJPO UHVSHFWLYHO\ The

potency of activity against AChE was BC > BE > LE > BH >LH > LC.

Only 4 compounds showed good to moderate enzyme inhibition namely

syringic acid (IC50 29.53±0.19 µg/ml), scopoletin (IC50 51.00±0.02 µg/ml),

scoparone (IC50 86.58±0.05 µg/ml) and vomifoliol (IC50 96.64±0.09

µg/ml). Rest of the compounds showed poor enzyme inhibition with IC50

values >100 µg/ml.

vii. Antimicrobial activity

All crude extracts of Canarium patentinervium Miq. under investigation

exhibited exceptional concentration-dependent antimicrobial activity

against both Gram-positive and Gram-negative bacteria. The most

promising activity was displayed against Gram-positive bacteria

Staphylococcus aureus, MRSA, Bacillus cereus and Gram-negative

bacteria and Pseudomonas aeruginosa, Klebsiella sp, Klebsiella

pneumoniae ESBL and yeast Candida parapsilopsis. The antimicrobial

activity exhibited by the bark extracts and ethanol extract of leaves against

MSSA and MRSA indicates exceptional antimicrobial activity. All the

tested bacterias were susceptible to either bark or leaf extracts.

318

All isolated compounds tested against Staphylococcus aureus ATCC

11632 showed bacterial growth inhibition. Scopoletin, scoparone, hyperin,

cynaroside and syringic acid had bactericidal effect <100 µg/ml. Only

scopoletin had bactericidal effect and complete kill at MBC 50.00 µg/ml.

viii. Antifungal activity

The most promising activity was displayed against yeast Candida

parasilopsis. The ethanol extracts and hexane extract of barks also showed

inhibition against Candida parasilopsis with a cidal and static action

respectively.

ix. Anticancer activity

The chloroform and ethanol extract of barks showed good antitumor

activities with GI50 values of 23.44±0.05 ȝJPODQG±0.21 ȝJPO7KH

most susceptible cell lines were found to be the breast cancer cell line,

MDA 468.

Scopoletin displayed potent anticancer effect against breast cancer cell line

MDA 468 (GI50 0.09±0.25 µg/ml) and colorectal cancer cell line HT-29

(GI50 0.17±0.05 µg/ml), the latter being more significant that positive

control doxorubicin (GI50 0.66±0.60 µg/ml).

x. Anti parasitic activity

The hexane extract of leaves showed moderate antileishmanial activity

with IC50 YDOXHVRIȝJPO7KLVFRXOGEHGXHWRHVVHQWLDORLOV

319

present in the hexane extracts as shown previously in the family of

Burseraceae [216]. Only ethanol extracts showed activity against Giardia

intestinalis and Entamoeba histolytica at concentration of 500 ȝJPO

Other extracts were inactive against these two intestinal parasites.

Scopoletin was tested against all three parasite amd it was more potent

against Leishmania donovani (IC50 163.30±0.32 µg/ml) and MIC of >200

µg/ml for both Giardia intestinalis and Entamoeba histolytica. Sensitivity

of Leishmania donovani is then followed by with the presence of lioxin

(IC50 211.48±0.32 µg/ml), vomifoliol (IC50 302.80±0.33 µg/ml), scoparone

(IC50 329.90±0.32 µg/ml) and (+)-catechin (IC50 478.93±0.28 µg/ml).

In Chinese folk medicine, Canarium album (Lour.) Raeusch has been used for

treatment of various diseases ranging from cardiovascular, gastrointestinal and

toxicities while Canarium schweinfurthii Engl. has been used by traditional healers as

a remedy for diabetes mellitus in southern Senegal while in Congo and Central

African Republic the plant is used in fever, as stimulant, emollient, in post-partum

pain, constipation, malaria, diarrhoea, sexual infections and rheumatism (Chapter 1)

to name a few. The fact only 12 % of 75 species of Canarium have been studied for

their pharmacological activities promises an unopened crypt of various secondary

metabolites as lead compounds and biological effects that needs to be uncovered and

investigated. Thus, by using an ethnopharmacological approach, the investigation of

Canarium patentinervium Miq. a plant from Malaysia that has been used by the orang

asli (indigenous people) for healing wounds was done. Crude extracts and isolated

320

secondary metabolites were investigated for their various activities.. Three out of

eight compounds were isolated for the first time from this genus of 75 species

(Canarium). Six kinds of major biological effects were evident in the crude and

compounds namely, antioxidant, antibacterial, anti-inflammatory, anti AChE, anti

parasitic, and anticancer all of which were reported for the first time from this plant.

This study is therefore a small leap in the field of an important endeavor for the

discovery of potent biologically active molecules for the treatment of various

diseases. This discovery also opens forth a pathway where biologically active

molecules isolated from biosynthesis of plants can now pose as lead molecules in the

field of synthetic chemistry for drug discovery.

As modern cultures and scientific advances spread around the world, the depth of the

knowledge store of traditional use still remains crucial. The full significance of the

indigenous knowledge forfeited may not be realised. It is thus important that the

knowledge be documented and the traditional use be given some credence through

modern scientific studies. Canarium patentinervium Miq. is such an example.

321

CHAPTER 13

RECOMMENDATIONS FOR FUTURE WORK

This study intends to contribute towards the knowledge base of plant species with

therapeutic potential. Material for this study was only collected from a single site. To

account for possible geographical and chemotypic variation material should be

studied from several populations.

i. Antioxidant activity

The isolated compounds should be tested for synergistic or antagonistic effect by the

construction of an isobologram.

ii. Antimicrobial activity

,VRODWLRQ DQG VWUXFWXUDO HOXFLGDWLRQ RI WKH FRPSRXQGV from the tannin fraction

responsible for the antimicrobial activity of Canarium patentinervium Miq. should be

investigated.

'HWHUPLQDWLRQRIV\QHUJLVWLFDQWDJRQLVWLFor additive effects should be investigated.

iii. Anti-inflammatory Activity

The actual efficacy of an anti-inflammatory activity needs to be validated by testing

the isolated bioactive compounds in a cell-based environment (in vitro) and in a

biological system (in vivo).

322

The effects of these active compounds on the suppression of the COX-2 gene

expression, requires investigation and clarification. The effectiveness of interaction of

these extracts and isolated compounds, including the flavonoid derivatives, with other

pro-inflammatory biochemical pathways may be assessed, and the possible structure-

activity relationships determined. This will provide a better understanding of the

possible mode of action.

iv. Anti AChE activity

More secondary metabolites should be isolated and tested in vitro as it may lead to

the development of novel treatments in the global struggle against neurodegenerative

diseases.

All the compounds should also be subjected to cell based study to investigate the

mechanism of actions.

v. Anticancer

7KLV VWXG\ KDV ZLWKRXW D Goubt, proven the existence of other compound or

compounds (besides scopoletin) with potential in vitro anticancer activity in different

species and extracts of Canarium patentinervium Miq. Activity-guided fractionation,

isolation and identification of these compounds is imperative and may lead to the

development of novel treatments in the global struggle against cancer and cancer-

related ailments.

vi. Antiparasitic activity

More secondary metabolites should be isolated and tested for the efficacy against

parasitic kill and it should be tested against various clinical strains of parasites.

323

vii. Toxicity

:KLOHLQGLJHQRXVCanarium patentinervium Miq. has edible fruit, it is important to

note that in order to establish a toxicity profile, other cell lines should be investigated

such as the liver HepG2 cells, as well as in vivo studies.

viii. Phytochemical investigation

5HVHDUFK RQ WKH QRQ-volatile compounds warrant further study. Further analysis

must be conducted using different detectors and chromatographic techniques such as

liquid chromatography ± mass spectrometry, to provide a greater insight of the

phytochemical composition of this species.

3ODQWV SURGXFH D ZLGH UDQge of secondary metabolites, which may prove to be

invaluable in development of drugs, flavours, fragrances, dyes, anti-oxidants and

insecticides. It is thus important to locate and determine the role of these secondary

metabolites in plants and unravel their biosynthesis.

Finding new leads for drug development, and determining the biosynthesis of such

products, thus requires a different approach which researchers are developing.

Metabolomics investigates the end products of cellular functions. The levels of these

metabolites are viewed as a response of the biological systems to environmental or

genetic manipulation. The use of metabolomics in plant studies will enable the

characterisation and differentiation of genotypes and phenotypes based on the levels

of metabolites, and also aid in the rapid screening of multiple extracts giving an

exceptionally broad overview of the chemistry. This may also provide a means of

improving the production of certain metabolites in plants through genetic engineering.

324

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356

APPENDIX A: NMR DATA

Figure A1: 1H NMR spectrum of compound 1

357

Figure A2: 13C NMR spectrum of compound 1

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Figure A13: HMBC spectrum of compound 7

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Figure A14: COSY spectrum of compound 7

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Fiigure A18: 13C NMR spectrum of compound 8

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APPENDIX B: HPLC CHROMATOGRAMS

Figure B1: HPLC chromatogram for compound 6

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376


377

APPENDIX C: PUBLICATIONS

SAGE-Hindawi Access to Research Biotechnology Research International Volume 2011, Article ID 768673, 5 pages doi:10.4061/2011/768673

Research Article In Vitro Antimicrobial, Antioxidant Activities and Phytochemical Analysis of Canarium patentinervium Miq. fromMalaysia R.Mogana,1 Khoo Teng-Jin,1 and C.Wiart2 1 School of Pharmacy, Faculty of Science, University of Nottingham, Malaysia Campus, JlnBroga, Semenyih, 43500 Selangor, Malaysia 2 School of Biomedical Science, Faculty of Science, University of Nottingham, Malaysia Campus, JlnBroga, Semenyih, 43500 Selangor, Malaysia Correspondence should be addressed to R. Mogana, [email protected] Received 3 March 2011; Revised 7 April 2011; Accepted 2 May 2011 Academic Editor: Goetz Laible Copyright © 2011 R. Mogana et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Six different extracts of Canarium patentinervium Miq. (Burseraceae) leaves and barks were screened for their phytochemical composition, and antimicrobial and free radical scavenging activities. Among the different extracts tested, the ethanol extract of leaves showed significant antimicrobial and radical scavenging activities. The most susceptible micro-organisms were found to be Gram-positive bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus or MRSA) and Gram-negative bacteria (Pseudomonas aeruginosa). Phytochemical analysis of the extracts revealed that the antimicrobial and the radical scavenging activities are mainly due to the presence of tannins and flavonoids. The results obtained suggest that Canarium patentinervium Miq. could be exploited in the management of various infectious diseases.

379

R.Mogana et al. / Journal of Pharmacy Research 2011,4(8),2482-2489

2482-2489

Review Article ISSN: 0974-6943

Available online through

www.jpronline.info

Canarium L. : A Phytochemical and Pharmacological Review

R.Mogana1* and C.Wiart2

1School of Pharmacy, Faculty of Science, University of Nottingham(Malaysia Campus), Jln Broga, Semenyih, 43500, Selangor Darul Ehsan, Malaysia

2School of Biomedical Science, Faculty of Science, University of Nottingham(Malaysia Campus), Jln Broga, Semenyih, 43500, Selangor Darul Ehsan, Malaysia

Received on: 17-05-2011; Revised on: 12-06-2011; Accepted on:16-07-2011

ABSTRACT The genus Canarium L. consists of 75 species of aromatic trees which are found in the rainforests of tropical Asia, Africa and the Pacific. The medicinal uses, botany, chemical constituents and pharmacological activities are now reviewed. Various compounds are tabulated according to their classes their structures are given. Traditionally Canarium L. species have been used to treat a broad array of illnesses. Pharmacological actions for Canarium L. as discussed in this review include antimicrobial, antioxidant, anti-inflammatory, hepatoprotective and antitumor activity.

Keywords: Canarium L., Burseraceae, antibacterial, antioxidant, pharmacology, secondary

metabolites

380

Planta Med 2011; 77 - PL9

DOI: 10.1055/s-0031-1282658

The first antibacterial activity report of three selected Malaysian

rainforest medicinal plants

A Nematollahi 1, N Aminimoghadamfarouj 1, M Rajagopal 1, TJ Khoo 1, C Wiart 2

1School of Pharmacy, Faculty of Science, Nottingham University, 43500, Malaysia

2School of Biomedical Science, Faculty of Science, Nottingham University, 43500, Malaysia

Despite increasing resistance among clinically important gram-negative and gram-positive pathogens

to many common antibacterial agents, many large pharmaceutical companies are showing decreased

interest in this product area that is so critical to public health. This downturn in antibacterial discovery

and development, in turn, is leaving us vulnerable to emerging resistance, particularly to recently

arrived vancomycin-resistant Staphylococcus aureus and multiply resistant, gram-negative bacilli for

which we do not have adequate antimicrobial therapy in the future. Rainforest plants possess

therapeutic potential, including antimicrobial activity [1,2]. Therefore, this study is a screening

program of several extracts from three endemic medicinal plants in Malaysian rainforest which have

not been fully discovered and investigated for their antimicrobial properties against several Gram-

positive and Gram-negative bacteria strains. These plants included families namely Annonaceae,

Ebenaceae and Burseraceae. The antibacterial activity of hexane, chloroform and ethanol fractions of

some parts of Uvaria grandiflora Roxb. (Annonaceae), Diospyros wallichii King & Gamble

(Ebenaceae) and Canarium patentinervium Miq. (Burseraceae) was determined against Gram-positive

bacteria Bacillus cereus ATCC10876, Staphylococcus aureus ATCC11632, Methicilin resistant

Staphylococcus aureus ATCC43300 and Gram- negative bacteria Pseudomonas aeruginosa

ATCC10145 and Escherichia coli ATCC10536 using the disk diffusion method. Results showed that

the bark ethanol fraction of U. grandiflora, the fruit hexane fraction of D. wallichii and the leaf ethanol

fraction of C.patentinervium are active (Table 1). The results indicate that these medicinal herbs can be

used as active and potent ingredients in the formulation of natural antibacterial products.

References:

1- Wiart C (2006) Medicinal Plants of the Asia-Pacific: Drugs from the Future, World Scientific,

Singapore.

2. Harvey AL (1999) Trends Pharmacol Sci 20: 196-198.

381

Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2013, Article ID 734824, 7 pages http://dx.doi.org/10.1155/2013/734824

Research Article Anti-Inflammatory, Anticholinesterase, and Antioxidant Potential of Scopoletin Isolated from Canarium patentinervium Miq. (Burseraceae Kunth) R. Mogana, K. Teng-Jin, and C.Wiart Center for Natural and Medicinal Products Research, School of Pharmacy, Faculty of Science, University of Nottingham (Malaysia Campus), Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia. Correspondence should be addressed to R. Mogana; [email protected] Received 13 March 2013; Accepted 14 June 2013 Academic Editor: Mohd Roslan Sulaiman Copyright © 2013 R. Mogana et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Bioassay guided fractionation of an ethanol extract of leaves of Canarium patentinervium Miq. (Burseraceae Kunth.) led to the isolation of scopoletin. The structure of this coumarin was elucidated based on spectroscopic methods including nuclear magnetic resonance (NMR-1D and 2D) and mass spectrometry. Scopoletin inhibited the enzymatic activity of 5-lipoxygenase and acetyl cholinesterase with an IC50 equal to 1.76 ± 0.01 ߤM and 0.27 ± 0.02mM, respectively, and confronted oxidation in the ABTS, DPPH, FRAP, and ߚ-carotene bleaching assay with EC50 values equal to 5.62 ± 0.03 ߤM, 0.19 ± 0.01 mM, 0.25 ± 0.03mM and 0.65 ± 0.07 mM, respectively. Given the aforementioned evidence, it is tempting to speculate that scopoletin represents an exciting scaffold from which to develop leads for treatment of neurodegenerative diseases.

382

Hindawi Publishing Corporation ISRN Biotechnology Volume 2013, Article ID 986361, 8 pages http://dx.doi.org/10.5402/2013/986361

Research Article The Medicinal Timber Canarium patentinervium Miq. (Burseraceae Kunth.) Is an Anti-Inflammatory Bioresource of Dual Inhibitors of Cyclooxygenase (COX) and 5-Lipoxygenase(5-LOX) R. Mogana, K. Teng-Jin, and C.Wiart Center for Natural and Medicinal Products Research, School of Pharmacy, Faculty of Science, University of Nottingham (Malaysia Campus), Jln Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia Correspondence should be addressed to R. Mogana; [email protected] Received 10 July 2013; Accepted 19 August 2013 Academic Editors: A. O. Ballesteros, A. Maggio, and D. Pant Copyright © 2013 R. Mogana et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The barks and leaves extracts of Canarium patentinerviumMiq. (Burseraceae Kunth.) were investigated for cyclooxygenase (COX) and 5-lipoxygenase (LOX) inhibition via in vitro models.The corresponding antioxidative power of the plant extract was also tested via nonenzyme and enzyme in vitro assays.The ethanolic extract of leaves inhibited the enzymatic activity of 5-LOX, COX-1, and COX-2 with IC50 equal to 49.66±0.02 ߤg/mL, 0.60±0.01 ߤg/mL, and 1.07±0.01 ߤg/mL, respectively, with selective COX-2 activity noted in ethanolic extract of barks with COX-1/COX-2 ratio of 1.22.The ethanol extract of barks confronted oxidation in the ABTS, DPPH, and FRAP assay with EC50 values equal to 0.93 ± 0.01 ߤg/mL, 2.33 ± 0.02 ߤg/mL, and 67.00 ± 0.32 ߤg/mL, respectively, while the ethanol extract of leaves confronted oxidation in ߚ-carotene bleaching assay and superoxide dismutase (SOD) assay with EC50 value of 6.04 ± 0.02 ߤg/mL and IC50 value of 3.05 ± 0.01 ߤg/mL. The ethanol extract acts as a dual inhibitor of LOX and COXenzymes with potent antioxidant capacity.The clinical significance of these data is quite clear that they support a role for Canarium patentinervium Miq. (Burseraceae Kunth.) as a source of lead compounds in the management of inflammatory diseases.

383

APPENDIX D: CONTINUOUS EDUCATION

x 2SWLPL]LQJ <RXU /DERUDWRU\¶V 3URGXFWLYLW\ YLD LPSURYHG 6DPSOH 3UHSDUDWLRQ

Technique and Advanced Column Technology, Monash University, 12th Oct

2010.

x Project Management for Research, Graduate School Training Course, Nottingham

University Malaysia Campus, 4th Oct 2010

x Getting Going on Your Thesis, Graduate School Training Course, Nottingham

University Malaysia Campus, 5th Oct 2010

x Lab Demonstrations (Science) , Graduate School Training Course, Nottingham

University Malaysia Campus, 17th Jan 2011.

x Back To Nature symposium, Prof Iqbal Choudhary, UITM Puncak Alam, Jan

2011.

x NMR Theory & Applications: Natural products, UITM Puncak Alam, 17-18th Feb

2011.

x HPLC column determination, UITM Puncak Alam, April 2011.

x Workshop 2011: NMR for Intermediates and Metabolomics, UITM Puncak Alam,

17-19th Nov 2011.

x Technical Writing and Getting Published, Graduate School Training Course,

Nottingham University Malaysia Campus, 14th March 2012.

x Finishing Your Thesis, Graduate School Training Course, Nottingham University

Malaysia Campus, 30th Nov 2012.

x NMR Spectroscopy Workshop, UiTM Puncak Alam, 1-3rd March 2013.

Rajagopal, Mogana Sundari (2014) Canarium patentinervium miq....

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